Isolated liver stem cells

ABSTRACT

Isolated liver progenitor stem cells and cell populations of isolated liver progenitor stem cells are disclosed. The progenitor stem cells originate from adult liver, especially human adult liver. The isolated progenitor stem cells have uses in medicine, hepatology, inborn errors of liver metabolism transplantation, infectious diseases and liver failure. Methods of isolating these cells and their culture is described. The isolated cells are characterized before and after differentiation. Their use for transplantation and as animal models of human disease, toxicology and pharmacology is disclosed.

FIELD OF THE INVENTION

The present invention relates to isolated liver progenitor or stemcells, originated from adult liver, and to their use in medicine,hepatology, inborn errors of liver metabolism, transplantation,infectious diseases, liver failure. The present invention also relatesto methods of isolating these cells, their culture, characterizationbefore and after differentiation, and their use for transplantation,animal models of human disease, artificial organ devices, toxicology andpharmacology.

BACKGROUND OF THE INVENTION

Liver is a key organ performing many vital functions such as glucosehomeostasis, xenobiotic detoxification or macromolecule synthesis.Hence, impairment of one of the multiple liver functions could have adramatic impact on health. Worldwide incidence of acute or chronic liverdiseases sets these pathologies between the 5th and the 9th cause ofdeath, according to the World Health Organization. So far, the onlycurative treatment for end-stage liver disease remains livertransplantation. The outcome for patients who went through surgicalliver replacement is rather good; with more than 95% of recovery.However, despite new surgical techniques, including split-liver andliving-related donor, the increasing organ shortage leads to highermortality on the waiting list. Therefore, an important goal intransplantation medicine research is the demonstration of potential useof liver cells in liver regeneration and treatment of hepatic diseases.

Liver cell transplantation (LCT) is an emerging procedure, involving theinfusion of liver cell suspension in the portal system of the recipient.It aims to a recovery of the recipient's liver function as a consequenceof engraftment and repopulation of the diseased parenchyma. LCT wasfirst validated in animal models in which syngeneic hepatocytes havebeen shown to survive indefinitely and be able to correct various enzymedefects (for review, see Najimi and Sokal. 2005. Minerva Pediatr 57(5):243-57).

In human, early studies were designed for the treatment of acute liverfailure. These studies prompted clinicians to extend LCT to furtherindications and so far at least thirty cases have been reportedworldwide for various defects (Strom et al. 1997. Transplant Proc 29(4):2103-6). In the specific field of metabolic diseases, thirteen casesreported the use of hepatocytes, for the treatment of, among others,Crigler-Najjar syndrome type I, urea cycle defects or rare diseases,such as, e.g., infantile Refsum's disease. These studies demonstratedthe engraftment of hepatocytes within the parenchyma and consequently animprovement of the patient status up to 18 months post-transplantation.

However, because supply of mature human hepatocytes for transplantationis still limited, in fact more or less as limited as the availability ofwhole liver, research also aims at obtaining transplantable cells fromother sources, such as progenitor and stem cells, e.g., of embryonic oradult origin, that could be expandable, e.g., in vitro, and able todifferentiate into functional mature hepatocytes, esp. in vivo aftertransplantation. Accordingly, there is a great need to develop new meansthat are useful in treating various diseases or conditions associatedwith liver associated diseases, particularly given the inadequatetreatments currently available for the majority of these disorders.

Historically, embryonic stem (ES) cells were thought to be involved onlyin the organogenesis, due to their observed unlimited clonal divisionand pluripotent differentiation into daughter cells of entire tissues.On the other hand, regeneration processes in adult organs were typicallyascribed to adult progenitor cells. Nevertheless, this theory has beenrevised in view of the discovery in adult organs of stem cellsexpressing known embryonic markers. Hence, characterizations of stem andprogenitor cells are now based not only on the development process(embryonic versus adult), but also on the presence of specific cellularmarkers therein. Indeed, expression of cellular markers, such asmembrane proteins or transcription factors, may vary along thedifferentiation pathways and reflect various stimuli (e.g.,environmental stimuli) and cellular needs. Often, it is observed that inthe course of a differentiation process, a stem cell will graduallycease to display markers indicative of its pluripotence, e.g., Oct-4,and being to express markers attributable to later stages, e.g., markersof a specific lineage. As a non-limiting example, Oct-4 may beprogressively lost through maturation and, on the other hand, cellsentering the endodermal lineage may begin to express alpha-fetoprotein.

Concerning liver regeneration through cell transplantation, severalpossible source cell types may be considered. For example, ES cellswould be expected to be capable of regenerating any organ, due to theirpluripotence. Indeed, this avenue is extensively explored in the art.However, ES cells are prone to generate tumour growth when introduced inany other tissue than in utero. Therefore, their use in vivo remainslimited by the risk of carcinogenic deviation. Even successful prior invitro differentiation of ES cells might not be safe enough to considerhuman inoculation.

A safer alternative would be the use of adult progenitor cells which,unlike ES cells, tend to display limited capacity for clonal divisionand their differentiation give rise to daughter cells with more limitedfates. In liver, adult progenitors such as oval cells (cholangiocytesand hepatocytes precursors) or small hepatocyte-like cells have beendescribed. However, their medical use is rendered difficult by theirscarcity in normal adult organs.

Consequently, adult stem cells that would show capability of clonaldivision with reduced or absent risk of carcinogenic deviation wouldrepresent a great improvement in cell transplantation sources. Severaltypes of adult stem cells are currently being evaluated in liver celltransplantation studies. For example, mesenchymal stem cells (MSC) fromperipheral or umbilical cord blood are being studied due to theirability to trans-differentiate into more mature cells from anotherlineage. Moreover, hematopoietic stem cells from marrow have also beenstudied in terms of liver regeneration potencies.

While in vitro characterization of adult stem cells still presentsdifficulties, it is currently accepted in the field that suchcharacterisation may advantageously involve detecting (i) markers of itsembryonic origin or lineage (esp., mesodermal, endodermal, ectodermal orhematopoietic), (ii) expression of markers reflecting the level ofdifferentiation and thus to some degree predictive of the differentpossible progenies and, (iii) in vitro or in vivo fate afterdifferentiation. Consequently, characterisation and distinguishing ofadult stem cells obtainable from normal liver may advantageously involveevaluation of the presence or absence of (i) marker(s) reflecting thecomplex embryonic origin of this organ, (ii) marker(s) ofdifferentiation (e.g., presence of albumin) and, (iii) at least onemarker indicative of the stem cell fate.

According to current knowledge, liver originates mainly from theendoderm and hepatocytes are part of the endodermal lineage. However,the formation of the hepatic cells also involves the interaction betweenthe endodermal epithelium and the cardiogenic mesoderm. Furthermore, infoetal development haematopoiesis also takes place in the liver. Giventhis interplay during development, one needs to be open minded whencontemplating markers present in adult liver stem cells, since markersof the endodermal, mesodermal and/or haematopoietic lineages might beexpected.

When assessing the differentiation level and cell type commitment,different cell markers may be evaluated, as carried out further in thisdisclosure. For example, during the differentiation process of cellssome markers may decrease or disappear, others may increase or may begained, and yet others may be maintained all way to a specialised andfunctional cell. By virtue of non-limiting example, duringorganogenesis, i.e. during foetal life, hepatoblasts are considered tobe the common progenitors of cells forming the parenchyma (esp.hepatocytes and biliary cells) and express inter alia cytokeratin-7(CK-7) as well as CK-19, albumin and α-fetoprotein. In adult liver, aknown common progenitor for hepatocytes and biliary cells is the ovalcell, that expresses CK-19, albumin and α-fetoprotein. Afterdifferentiation into biliary cells, expression of CK-19 andα-fetoprotein is maintained while expression of CK-7 (considered afeature of more immature cells) tends to cease. On the other hand,hepatocytes maintain expression of α-fetoprotein and albumin, but do notshow expression of the above CK. Also from this example, it follows thatstem cell characterization may be complex, but that the assessment ofmarkers may be advantageously used to indicate the cells' type orproperties.

To the inventors' knowledge, previous studies described the isolation ofprogenitor cells from normal adult liver, which cells demonstrated morethan one cell fate. An adult liver stem cell line capable of in vitroamplification and in vivo differentiation into hepatocytes, andpreferably with only the hepatocytic cell fate has not been described.Moreover, previous studies used complicated techniques, such as FACS,calcium-implemented media or specific density gradients to isolate theirliver stem cells.

Accordingly, it is an object of the invention to provide novel adultliver derived progenitor or stem cells with improved properties, andparticularly useful in, e.g., liver cell transplantation. The inventionalso sets out to provide a simple method to isolate the said cells.

SUMMARY OF THE INVENTION

The invention provides adult liver derived progenitor or stem cells,cell lines thereof or cell populations comprising such, obtained from anormal liver tissue. Methods of isolating these cells, their culture,characterization before and after differentiation, and their use fortransplantation, animal models of human disease, toxicology andpharmacology are also within the invention.

In an aspect, the present invention realised a novel, isolatedprogenitor or stem cell (a vertebrate, preferably a mammal, even morepreferably a human cell), originated from adult liver, characterised inthat it co-expresses (i.e., is positive for) at least one mesenchymalmarker, esp. one, more than one, e.g., 2, 3, or 4, or all of the markersCD90, CD44, CD73, vimentin and α-smooth muscle actin (ASMA), with thehepatocyte marker albumin (ALB) and possibly with one or more otherhepatic or hepatocyte markers, preferably one, more than one, or allCD29, alpha-fetoprotein (AFP), alpha-1 antitrypsin and/or MRP2transporter. The said adult liver progenitor or stem cell may furtherexpress one, more than one, or all of the following molecules indicativeof hepatocyte-like properties or function: G6P, CYP1B1, CYP3A4, HNF-4,TDO, TAT, GS, GGT, CK8, EAAT2. The said adult liver progenitor or stemcell may further be characterised by one, more than one, or all of thefollowing: negative for at least the hematopoietic markers CD45 and CD34and possibly also for one or more other hematopoietic markers, such as,e.g., CD105, HLA-DR, negative for the cholangiocyte epithelial markercytokeratin-19 (CK-19) and possibly for more epithelial markers;negative for at least the undifferentiated stem cell markers CD117 andOct-4 and possibly also for one or more than one embryonic stem cellmarkers; low level expression of AFP. Preferably, the said adult liverprogenitor or stem cell may have mesenchymal-like morphology, inparticular involving one, more than one or all of growth in monolayers,flattened form, broad cytoplasm and/or ovoid nuclei with one or twonucleoli.

In an embodiment, in particular the present invention provides anisolated stem cell originated from adult liver, which is CD90, CD29 andCD44 positive which is albumin-positive, vimentin-positive and alphasmooth muscle actin-positive. In an embodiment, the isolated stem cellsare also CK-19 negative, CD45 negative, CD 34 negative and CD117negative. The present invention also provides a cell populationcomprising progenitor mesenchymal stem cells having at least three ofthe following characteristics: antibody-detectable expression ofalbumin; antibody-detectable expression of vimentin; antibody-detectableexpression of alpha smooth muscle actin; absence of CK-19; absence ofCD45, absence of CD45 marker, absence of CD34 marker, absence of CD117marker, evidence of CD90 marker; evidence of CD29 marker; or evidence ofCD44 marker. Preferably the cells have all the above listedcharacteristics. In a preferred embodiment, the stem cells are humanliver stem cells.

In an embodiment, the isolated stem cell originated from adult liver isCD90, CD73, CD29 and CD44 positive and is albumin-positive,vimentin-positive and alpha smooth muscle actin-positive.

The invention also provides a method for obtaining an isolatedprogenitor or stem cell or a cell population comprising the saidprogenitor or stem cell, the method comprising: (a) disassociating adultliver or a part thereof to form a population of primary cells from thesaid adult liver or part thereof, (b) plating the primary cellpopulation onto a substrate which allows adherence of cells thereto, and(c) culturing cells from the primary cell population, which have adheredto the said substrate, for at least 7 days, preferably at least 10, atleast 13, or at least 15 days.

The present invention also provides a method for obtaining the isolatedliver stem cell or the population thereof according to the inventioncomprising the steps of culturing cells from adult liver, isolatinghepatocytes therefrom, plating the hepatocytes and culturing saidhepatocytes for at least 7 days, preferably at least 10, at least 13, atleast 15 days.

In a yet further aspect, the invention provides an isolated adult liverprogenitor or stem cell, cell line thereof and/or a cell populationcomprising such, obtainable by or directly obtained using the methods ofthe invention.

In a further aspect, the present inventors have established a particularcell population (cell line) of adult human liver progenitor or stemcells according to the invention and deposited the said isolated cellline on Feb. 20, 2006 under the Budapest Treaty with the BelgianCoordinated Collections of Microorganisms (BCCM/LMBP) under accessionnumber LMBP 6452CB (given by the International Depositary Authority;identification reference given by the depositor: ADHLSC). Accordingly,the present invention relates to an isolated cell, cell line and cellpopulation deposited with BCCM under accession number LMBP 6452CB(herein, the “LMBP 6452CB” cell line), sub-lines thereof includingclonal sub-lines, and to progeny thereof, including differentiatedprogeny thereof, esp. hepatocytes or hepatocytes-like cells preparedtherefrom, and to genetically or otherwise modified derivatives thereof.

The present invention also provides a composition comprising theisolated liver progenitor or stem cell or the population thereofaccording to the invention. Preferably, the liver cells are human livercells, or mammalian liver cells.

The progenitor or stem cells according to the invention (specificallymentioning, albeit of course not limited to the LMBP 6452CB line)entails several considerable advantages. For example, unlike cells ofembryonic origin, the present progenitor or stem cells are of adultorigin and may display lower risk of uncontrolled (tumour) growth ormalignant transformation when used in therapy.

Furthermore, the inventors realised that the progenitor or stem cellsaccording to the invention substantially do not display ability todifferentiate into mesodermal cell types (e.g., osteocytes orchondrocytes, connective tissue cells), which decreases ectopicformation of such tissues when the cells are administered and implantedin liver tissue.

The present inventors also realised that the progenitor or stem cells ofthe invention may have particular preference for differentiation tohepatocytes or hepatocyte-like cells, which makes them particularlysuitable for reconstitution of hepatocyte functions in a liver.

The present progenitor or stem cells are clearly different frompreviously described liver-derived stem cells, such as oval cells, e.g.,in their morphological characteristics and marker expression.

The adult liver progenitor or stem cells according to the invention areparticularly useful in medicine, hepatology, inborn errors of livermetabolism, transplantation, infectious diseases, liver failure. Theliver progenitor or stem cells according to the invention areparticularly useful for (human) liver cell transplantation, thepreparation of animal models of human liver cell transplantation,bio-artificial livers, in vitro liver cell lines and animal models ofacquired human liver diseases, liver metabolism screening tests(pharmacokinetics, cytotoxicity, genotoxicity) and liver cell directedgene therapy. The liver progenitor or stem cell according to theinvention can be further differentiated into hepatocytes.

The present invention also provides a pharmaceutical compositioncomprising a liver progenitor or stem cell, cell line thereof or cellpopulation thereof, or progeny thereof including differentiated progeny,esp. hepatocytes or hepatocyte-like cells, optionally geneticallymodified, according to the invention and a pharmaceutically acceptablecarrier. Preferably, the liver cells are human liver cells, or mammalianliver cells.

The present invention also provides a method of treating liver diseasecomprising administering an effective amount of a liver progenitor orstem cell, a cell line thereof or cell population thereof, or progenythereof including differentiated progeny, esp. hepatocytes orhepatocyte-like cells, optionally genetically modified, according to theinvention. In an embodiment, the liver disease includes but is notlimited to phenylketonuria and other aminoacidopathies, haemophilia andother clotting factor deficiencies, familial hypercholesterolemia andother lipid metabolism disorders, urea cycle disorders, glycogenosis,galactosemia, fructosemia, tyrosinemia, protein and carbohydratemetabolism deficiencies, organic aciduria, mitochondrial diseases,peroxysomal and lysosomal disorders, protein synthesis abnormalities,defects of liver cell transporters, defect of glycosylation, hepatitis,cirrhosis, inborn errors of metabolism, acute liver failure, acute liverinfections, acute chemical toxicity, chronic liver failure, cholangitis,biliary cirrhosis, Alagille syndrome, alpha-1-antitrypsin deficiency,autoimmune hepatitis, biliary atresia, cancer of the liver, cysticdisease of the liver, fatty liver, galactosemia, gallstones, Gilbert'ssyndrome, hemochromatosis, hepatitis A, hepatitis B, hepatitis C, andother hepatitis viral infections, porphyria, primary sclerosingcholangitis, Reye's syndrome, sarcoidosis, tyrosinemia, type 1 glycogenstorage disease, or Wilson's disease.

The present invention also provides a method for treating errors of geneexpression comprising: (i) introducing into a liver progenitor or stemcell, cell line thereof or cell population thereof, or progeny thereofincluding differentiated progeny, esp. hepatocytes or hepatocyte-likecells, according to the invention a functional copy of a gene to providea transformed population; and (ii) introducing into a patient's liver,which patient is in need of the functional copy of the gene, at least aportion of the transformed population. Alternatively, the transformedpopulation can be introduced into a non-human mammal's liver in order toproduce a new animal model of hepatic pathology.

The present invention also provides a composition for treating errors ofgene expression comprising a transformed liver progenitor or stem cell,cell line thereof or cell population thereof, or progeny thereofincluding differentiated progeny, esp. hepatocytes or hepatocyte-likecells, optionally genetically modified, according to the invention intowhich a functional copy of a gene has been introduced.

The present invention also provides a pharmaceutical composition fortreating errors of gene expression comprising a liver progenitor or stemcell, cell line thereof or cell population thereof, or progeny thereofincluding differentiated progeny, esp. hepatocytes or hepatocyte-likecells, according to the invention into which a functional copy of a genehas been introduced and a pharmaceutically acceptable carrier.

The present invention also provides a method for enhancing theregeneration of an injured or diseased liver comprising administeringinto the liver an effective amount of a liver progenitor or stem cell,cell lines thereof or cell population thereof, or progeny thereofincluding differentiated progeny, esp. hepatocytes or hepatocyte-likecells, optionally genetically modified, according to the invention.

The present invention also provides a liver assist device comprising ahousing harbouring a liver progenitor or stem cell, cell line thereof orcell population thereof, or progeny thereof including differentiatedprogeny, esp. hepatocytes or hepatocyte-like cells, optionallygenetically modified, according to the invention.

The present invention also provides a method of conducting in vitrotoxicity testing comprising: exposing to a test agent a liver progenitoror stem cell, a cell line thereof or cell population thereof, or progenythereof including differentiated progeny, esp. hepatocytes orhepatocyte-like cells, optionally genetically modified, according to theinvention, and observing at least one effect, if any, of the test agenton the population of liver cells. Preferably, the at least one effectincludes an effect on cell viability, cell function, or both.

The present invention also provides a method of conducting in vitro drugmetabolism studies comprising: (i) exposing a liver progenitor or stemcell, cell line thereof or cell population thereof, or progeny thereofincluding differentiated progeny, esp. hepatocytes or hepatocyte-likecells, optionally genetically modified, according to the invention, to atest agent, and (ii) observing at least one change, if any, involvingthe test agent after a predetermined test period. Preferably, the atleast one change includes a change in the structure, concentration, orboth of the test agent.

The present invention also provides a method of conducting testing forefficacious agents for treating liver infections comprising (i)infecting with an infectious agent of interest a liver progenitor orstem cell, cell line thereof or cell population thereof, or progenythereof including differentiated progeny, esp. hepatocytes orhepatocyte-like cells, optionally genetically modified, according to theinvention to provide an infected population, (ii) exposing the infectedpopulation to a predetermined amount of test agent, and (iii) observingeffects, if any, of the exposure on the infected population. In anembodiment, the infectious agent includes a microorganism. In anotherembodiment, the infectious agent includes one or more viruses, bacteria,fungi, or combinations thereof. In a particular embodiment, the observedeffects include effects on viral replication of a viral infectiousagent. Preferably, the viral infectious agent includes a hepatitisvirus.

The present invention also provides a method of producing a protein ofinterest comprising (i) introducing into a liver progenitor or stemcell, cell line thereof or cell population thereof, or progeny thereofincluding differentiated progeny, esp. hepatocytes or hepatocyte-likecells, according to the invention a functional gene encoding a proteinof interest, (ii) incubating the said cell population under conditionseffective for transcription, translation, and optionallypost-translational modification to take place, and (iii) harvesting theprotein of interest. Preferably the liver cells are human liver cells.In an embodiment, the protein of interest comprises a vaccine antigen.

The present invention also provides a method of conducting in vitro orin vivo studies on liver development and hepatocyte differentiationcomprising: exposing a liver progenitor or stem cell, a cell linethereof or cell population thereof, or progeny thereof includingdifferentiated progeny, according to the invention, in vitro or in vivoto conditions that affect differentiation conditions and observing atleast one effect on the population of the cells.

The present invention encompasses the cells, the preparation thereof,the characterization thereof, the culture method thereof and theproduction thereof.

The present invention also encompasses the preservation of these cellsby cryopreservation and the culture of the cells.

The present invention also encompasses the technique of transplantationof the cells in animals and humans.

The present invention also encompasses the use of the liver stem cellaccording to the invention for the preparation of a medicament for thetreatment of the above mentioned diseases. The present invention alsoencompasses the use of the liver stem cell according to the invention ina kit or kit in part.

BRIEF DESCRIPTIONS OF THE FIGURES

FIG. 1 shows the morphological appearance of human adult liver derivedprogenitor or stem cells of the invention (ADHLSC), as prepared inexample 1, after 1-month culture, using light microscopy (phasecontrast). A, at lower confluence; B, at higher confluence.Magnification 100×.

FIG. 2 A: presence of immuno-fluorescence staining of human adult liverderived progenitor or stem cells of the invention (ADHLSC), as preparedin example 1, after 1-month culture, for alpha-smooth muscle actin (A1),vimentin (A2) and albumin (A3, polyclonal, A4, monoclonal), B: RT-PCRgene expression profiles in the said (ADHLSC, lanes 1) cell line,compared to human hepatocytes (hHep, lanes 2), human stellate cells(hSC, lanes 3) and human hepatoblastoma (HepG2, lanes 4).

FIG. 3 shows differentiation of human adult liver derived progenitor orstem cells of the invention (ADHLSC) into hepatocytes-like lineage invitro. Cells were differentiated as in example 1, photographs were takenon day 2 (J2), day 14 (J14) and day 30 (J30) of the process.

FIG. 4 shows several images of hematoxylin and eosin staining inchimeric liver of uPA/SCID mice transplanted with undifferentiatedADHLSC cells, as detailed in example 1, demonstrating that these cellsbecome differentiated hepatocytes.

FIG. 5 shows several images of staining for human albumin unveiling thehuman origin of hepatocytes like population observed 10 weekspost-transplantation as in example 1 with undifferentiated ADHLSC.

DETAILED DESCRIPTION

As used herein, the singular forms “a”, “an”, and “the” include bothsingular and plural referents unless the context clearly dictatesotherwise. By way of example, “a cell” refers to one or more than onecell.

The terms “comprising”, “comprises” and “comprised of” as used hereinare synonymous with “including”, “includes” or “containing”, “contains”,and are inclusive or open-ended and do not exclude additional,non-recited members, elements or method steps.

All references cited in the present specification are herebyincorporated by reference in their entirety. In particular, theteachings of all references herein specifically referred to areincorporated by reference.

Obtaining Cells of the Invention

In an aspect, the present invention provides a method for obtaining anisolated progenitor or stem cell or a cell population comprising thesaid progenitor or stem cell, the method comprising: (a) disassociatingadult liver or a part thereof to form a population of primary cells fromthe said adult liver or part thereof, (b) plating the primary cellpopulation onto a substrate which allows adherence of cells thereto, and(c) culturing cells from the primary cell population, which have adheredto the said substrate, for at least 7 days, preferably at least 10, atleast 13, or at least 15 days.

As used herein, the term “isolated cell” refers generally to a cell thatis not associated with one or more cells or one or more cellularcomponents with which the cell is associated in vivo. For example, anisolated cell may have been removed from its native environment, or mayresult from propagation, e.g., ex vivo propagation, of a cell that hasbeen removed from its native environment.

The term “in vitro” as used herein denotes outside, or external to,animal or human body. The term “in vitro” as used herein should beunderstood to include “ex vivo”. The term “ex vivo” typically refers totissues or cells removed from an animal or human body and maintained orpropagated outside the body, e.g., in a culture vessel.

The term “cell population” refers generally to a grouping of cells.Unless indicated otherwise, the term refers to a cell groupingconsisting of or comprising isolated cells as defined herein.

A cell population may consist of cells having a common phenotype or maycomprise at least a fraction of cells having a common phenotype. Cellsare said to have a common phenotype when they are substantially similaror identical in one or more demonstrable characteristics, including butnot limited to morphological appearance, the presence, absence or levelof expression of particular cellular components or products, e.g., RNA,proteins or other substances, activity of certain biochemical pathways,proliferation capacity and/or kinetics, differentiation potential and/orresponse to differentiation signals or behaviour during in vitrocultivation (e.g., adherence, non-adherence, monolayer growth,proliferation kinetics, or the like). Such demonstrable characteristicsmay therefore define a cell population or a fraction thereof.

When a cell population is said herein to be “heterogeneous”, thisgenerally denotes a cell population comprising two or more cells orfractions of cells not having a common phenotype, e.g., a cellpopulation comprising cells of two or more different cell types. Bymeans of example and not limitation, a heterogeneous cell population canbe isolated from liver, and may comprise diverse liver cell types,including but not limited to hepatocytes (e.g., large and smallhepatocytes), cholangiocytes, Kupffer cells, hepatic stellate cells (Itocells) and liver endothelial cells.

When a cell population is said herein to be “homogeneous”, it consistsof cells having a common phenotype. A cell population said herein to be“substantially homogeneous” comprises a substantial majority of cellshaving a common phenotype. A “substantially homogeneous” cell populationmay comprise at least 70%, e.g., at least 80%, preferably at least 90%,e.g., at least 95%, or even at least 99% of cells having a commonphenotype, such as the phenotype specifically referred to (e.g., thephenotype of progenitor cell or stem cell). As used herein, the term“substantially homogeneous” as used herein may thus also encompass ahomogeneous population.

The term “cell population comprising a progenitor or stem cell” refersto a cell population as defined herein comprising at least oneprogenitor or stem cell and typically a fraction of progenitor cells orstem cells, as defined herein. Usually, the progenitor or stem cells ofthe said fraction may have a common phenotype.

The term “progenitor cell” refers generally to an unspecialised orrelatively less specialised and proliferation-competent cell, which orthe progeny of which can give rise to at least one relatively morespecialised cell type. By means of example and not limitation, aprogenitor cell may give rise to descendants that can differentiatealong one or more lineages to produce increasingly relatively morespecialised cells, wherein such descendants and/or increasinglyrelatively more specialised cells may themselves be progenitor cells, oreven to produce terminally differentiated cells, i.e., fully specialisedcells, which may be post-mitotic. The term also encompasses stem cellsare defined herein.

A progenitor cell is said to “give rise” to another, relatively morespecialised cell when, by means of example and not limitation, theprogenitor cell differentiates to become the other cell without firstundergoing cell division, or the other cell is produced after one ormore rounds of cell division and/or differentiation of the progenitorcell or progeny thereof.

The term “stem cell” refers to a progenitor cell capable ofself-renewal, i.e., can proliferate without differentiation, whereby theprogeny of a stem cell or at least part thereof substantially retainsthe unspecialized or relatively less specialised phenotype, thedifferentiation potential, and the proliferation competence of themother stem cell. The term encompasses stem cells capable ofsubstantially unlimited self-renewal, i.e., wherein the capacity of theprogeny or part thereof for further proliferation is not substantiallyreduced compared to the mother cell, as well as stem cells which displaylimited self-renewal, i.e., wherein the capacity of the progeny or partthereof for further proliferation is demonstrably reduced compared tothe mother cell.

A skilled person knows that the above properties generally refer to thein vivo behaviour of progenitor and stem cells, and may underappropriate conditions be completely or at least in part replicated invitro and/or ex vivo.

Based on the ability to give rise to diverse cell types, a progenitor orstem cell may be usually described as totipotent, pluripotent,multipotent or unipotent. A single “totipotent” cell is defined as beingcapable of growing, i.e. developing, into an entire organism. A“pluripotent” cell is not able of growing into an entire organism, butis capable of giving rise to cell types originating from all three germlayers, i.e., mesoderm, endoderm, and ectoderm, and may be capable ofgiving rise to all cell types of an organism. A “multipotent” cell iscapable of giving rise to at least one cell type from each of two ormore different organs or tissues of an organism, wherein the said celltypes may originate from the same or from different germ layers, but isnot capable of giving rise to all cell types of an organism. A“unipotent” cell is capable of differentiating to cells of only one celllineage.

The terms “differentiation”, “differentiating” or derivatives thereof asused herein denote the process by which an unspecialised or a relativelyless specialised cell becomes relatively more specialised. In thecontext of cell ontogeny, the adjective “differentiated” is a relativeterm. Hence, a “differentiated cell” is a cell that has progressedfurther down a certain developmental pathway than the cell it is beingcompared with. A differentiated cell may, for example, be a terminallydifferentiated cell, i.e., a fully specialised cell that takes upspecialised functions in various tissues and organs of an organism, andwhich may but need not be post-mitotic. In another example, adifferentiated cell may also be a progenitor cell within adifferentiation lineage, which can further proliferate and/ordifferentiate. Similarly, a cell is “relatively more specialised” if ithas progressed further down a certain developmental pathway than thecell it is being compared with, wherein the latter is thereforeconsidered “unspecialised” or “relatively less specialised”. Arelatively more specialised cell may differ from the unspecialised orrelatively less specialised cell in one or more demonstrable phenotypiccharacteristics, such as, for example, the presence, absence or level ofexpression of particular cellular components or products, e.g., RNA,proteins or other substances, activity of certain biochemical pathways,morphological appearance, proliferation capacity and/or kinetics,differentiation potential and/or response to differentiation signals,etc., wherein such characteristics signify the progression of therelatively more specialised cell further along the said developmentalpathway.

Non-limiting examples of differentiation may include, e.g., the changeof a pluripotent stem cell into a given type of multipotent progenitoror stem cell, the change of a multipotent progenitor or stem cell into agiven type of unipotent progenitor or stem cell, or the change of aunipotent progenitor or stem cell to more specialised cell types or toterminally specialised cells within a given cell lineage.Differentiation of an unspecialised or less specialised cell to a morespecialised cell may proceed through appearance of cells with anintermediate degree of specialisation.

Disassociating Liver Tissue

As mentioned, the method of the invention comprises a step ofdisassociating adult liver or a part thereof to form a population ofprimary cells from the said adult liver or part thereof.

The term “liver” refers to liver organ. The term “part of liver”generally refers to any part of the liver organ, without any limitationas to the quantity of the said part or the region of the liver organwhere it originates. Preferably, all cell types present in the liverorgan may also be represented in the said part of liver. Quantity of thepart of liver may at least in part follow from practical considerations,e.g., the need to obtain enough primary liver cells for reasonablypracticing the method of the invention. Such considerations will beapparent to a skilled person in view of the present teachings. Hence, bymeans of example and not limitation, a part of liver may represent(typically w/w) at least 0.1% of the liver, or at least 1%, or at least10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%,or at least 60%, or at least 70%, or at least 80%, or at least 90% ormore of the liver organ. In other non-limiting examples, a part of livermay be at least 1 g, or at least 10 g, or at least 100 g, or at least200 g, or at least 300 g, or at least 400 g, or at least 500 g, or atleast 600 g, or at least 700 g, or at least 800 g, or at least 900 g, orat least 1000 g, or at least 1100 g, or at least 1200 g, or at least1300 g, or at least 1400 g or more. For example, a part of liver may bea liver lobe, e.g., the right lobe or left lobe, or segment IV resectedduring split liver operation.

The term “adult liver” as used herein refers to liver which has attainedsubstantial developmental maturity of tissue organisation and cellcomposition.

In particular, it is known to a skilled person that liver may undergodevelopmental changes during a time period immediately following birth,where after it attains a substantially mature organisation. For example,in human subjects, the liver at birth contains a considerable populationof hematopoietic cells, which substantially disappear from the liverwithin about 1-2 weeks after birth. Moreover, the liver of humansubjects at birth contains a population of hepatic progenitor cells,which are substantially replaced by mature hepatocytes and biliary cellswithin several months after birth.

Accordingly, in human subjects, “adult liver” refers to liver ofsubjects that are any time after birth, preferably full term, and maybe, e.g., at least one month of age after birth, e.g., at least 2months, at least 3 months, e.g., at least 4 months, at least 5 months,e.g., at least 6 months age after birth, such as, for example, 1 year ormore, 5 years or more, at least 10 years or more, 15 years or more, 20years or more, or 25 years or more of age after birth. Hence, an “adultliver”, or mature liver, may be found in human subjects which wouldotherwise be described in the conventional terms of “infant”, “child”,“youth”, “adolescent” or “adult”.

A skilled person will appreciate that the liver may attain substantialdevelopmental maturity in different time postnatal intervals indifferent animal species, and can properly construe the term “adultliver” with reference to each species.

The liver or part thereof is obtained from a “subject”, “donor subject”or “donor”, interchangeably referring to a vertebrate animal, preferablya mammal, more preferably a human.

The term “mammal” includes any animal classified as such, including, butnot limited to, humans, domestic and farm animals, zoo animals, sportanimals, pet animals, companion animals and experimental animals, suchas, for example, mice, rats, rabbits, dogs, cats, cows, horses, pigs andprimates, e.g., monkeys and apes.

In a particularly preferred embodiment, the adult liver or part thereofis from a human subject. As detailed elsewhere in the specification,progenitor or stem cells or cell lines, or progeny thereof, derivedaccording to the invention from livers of human subjects, can beadvantageously used, e.g., in research and in therapy of patients, esp.human patients, suffering from liver disease.

In another embodiment, the adult liver or part thereof may be from anon-human animal subject, preferably a non-human mammal subject.Progenitor or stem cells or cell lines, or progeny thereof, derivedaccording to the invention from livers of non-human animal or non-humanmammal subjects can be advantageously used, e.g., in research and in thetherapy of liver disease in members of the same, related or othernon-human animal or non-human mammal species, or even in the therapy ofhuman patients suffering from liver disease (e.g., xenotransplantation,bio-artificial liver devices comprising non-human animal or non-humanmammal cells). By means of example and not limitation, particularlysuitable non-human mammal cells for use in human therapy may originatefrom pigs.

A donor subject may be living or dead, as determined by art-acceptedcriteria, such as, for example, the “heart-lung” criteria (usuallyinvolving an irreversible cessation of circulatory and respiratoryfunctions) or the “brain death” criteria (usually involving anirreversible cessation of all functions of the entire brain, includingthe brainstem). Harvesting may involve procedures known in the art, suchas, for example, biopsy, resection or excision.

A skilled person will appreciate that at least some aspects ofharvesting liver or part thereof from donor subjects may be subject torespective legal and ethical norms. By means of example and notlimitation, harvesting of liver tissue from a living human donor mayneed to be compatible with sustenance of further life of the donor.Accordingly, only a part of liver may typically be removed from a livinghuman donor, e.g., using biopsy or resection, such that an adequatelevel of physiological liver functions is maintained in the donor. Onthe other hand, harvesting of liver or part thereof from a non-humananimal may, but need not be compatible with further survival of thenon-human animal. For example, the non-human animal may be humanelyculled after harvesting of the tissue. These and analogousconsiderations will be apparent to a skilled person and reflect legaland ethical standards and are substantially not related to the essenceof the invention.

In an embodiment, liver or part thereof may be obtained from a donor,esp. human donor, who has sustained circulation, e.g., a beating heart,and sustained respiratory functions, e.g., breathing lungs or artificialventilation. Subject to ethical and legal norms, the donor may need tobe or need not be brain dead (e.g., removal of entire liver or portionthereof, which would not be compatible with further survival of a humandonor, may be allowed in brain dead human beings). Harvesting of liveror part thereof from such donors is advantageous, since the tissue doesnot suffer substantial anoxia (lack of oxygenation), which usuallyresults from ischemia (cessation of circulation).

In another embodiment, and as surprisingly realised by the presentinventors, liver or part thereof may be obtained from a donor, esp.human donor, who at the time of harvesting the tissue has ceasedcirculation, e.g., has a non-beating heart, and/or has ceasedrespiratory functions, e.g., has non-breathing lungs and no artificialventilation. While liver or part thereof from these donors may havesuffered at least some degree of anoxia, the present inventors realisedthat viable progenitor or stem cells according to the present inventioncan also be obtained from such tissues. Liver or part thereof may beharvested within about 24 h after the donor's circulation (e.g.,heart-beat) ceased, e.g., within about 20 h, e.g., within about 16 h,more preferably within about 12 h, e.g., within about 8 h, even morepreferably within about 6 h, e.g., within about 5 h, within about 4 h orwithin about 3 h, yet more preferably within about 2 h, and mostpreferably within about 1 h, such as, within about 45, 30, or 15 minutesafter the donor's circulation (e.g., heart-beat) ceased.

The tissues harvested as above may be cooled to about room temperature,or to a temperature lower than room temperature, but usually freezing ofthe tissue or parts thereof is avoided, esp. where such freezing wouldresult in nucleation or ice crystal growth. For example, the tissue maybe kept at any temperature between about 1° C. and room temperature,between about 2° C. and room temperature, between about 3° C. and roomtemperature or between about 4° C. and room temperature, and may beadvantageously be kept at about 4° C. The tissue may also be kept “onice” as known in the art. The tissue may be cooled for all or part ofthe ischemic time, i.e., the time after cessation of circulation in thedonor. That is, the tissue can be subjected to warm ischemia, coldischemia, or a combination of warm and cold ischemia. The harvestedtissue may be so kept for, e.g., up to 48 h before processing,preferably for less than 24 h, e.g., less than 16 h, more preferably forless than 12 h, e.g., less than 10 h, less than 6 h, less than 3 h, lessthan 2 h or less than 1 h.

The harvested tissue may advantageously be but need not be kept in,e.g., completely or at least partly submerged in, a suitable mediumand/or may be but need not be perfused with the suitable medium, beforefurther processing of the tissue. A skilled person is able to select asuitable medium which can support the survival of the cells of thetissue during the period before processing.

The method of the invention comprises disassociating adult liver tissueas described above to form a population of primary cells.

The term “disassociating” as used herein generally refers to partly orcompletely disrupting the cellular organisation of a tissue or organ,i.e., partly or completely disrupting the association between cells andcellular components of a tissue or organ. As can be understood by askilled person, the aim of disassociating a tissue or organ is to obtaina suspension of cells (a cell population) from the said tissue or organ.The suspension may comprise solitary or single cells, as well as cellsphysically attached to form clusters or clumps of two or more cells.Disassociating preferably does not cause or causes as small as possiblereduction in cell viability.

A suitable method for disassociating liver or part thereof to obtain apopulation (suspension) of primary cells therefrom may be any methodwell known in the art, including but not limited to, enzymaticdigestion, mechanical separation, filtration, centrifugation andcombinations thereof. In an embodiment, the method for disassociatingliver or part thereof may comprise enzymatic digestion of the livertissue to release liver cells. In an embodiment, the method fordisassociating liver or part thereof may comprise mechanical disruptionor separation of the liver tissue to release liver cells. In anembodiment, the method for disassociating liver or part thereof maycomprise a combination of enzymatic digestion and mechanical disruptionor separation of the liver tissue to release liver cells.

Methods for disassociating liver or part thereof as above are documentedin the art. For example, isolation of liver cells from liver tissue hasbeen well known since the mid-1960s (Howard et al. 1967. J Cell Biol 35:675-84). Rat hepatocytes were isolated using a combined mechanical andenzymatic digestion technique, subsequently modified by Berry and Friend(J Cell Biol 43: 506-20, 1969). This technique was further developed bySeglen to become the widely used two-step collagenase perfusiontechnique (Methods Cell Biol 13: 29-83, 1976).

Accordingly in an embodiment, the method for disassociating liver orpart thereof to obtain a population (suspension) of primary cellstherefrom is or comprises two-step collagenase perfusion technique. Askilled person is aware that since the above publication of the saidtechnique, various modifications thereof have been described and/or areconceivable, and are included in the invention.

By means of illustration and not limitation, brief description of acommon two-step collagenase technique ensues. For whole livers, cannulaecan be placed in the existing major blood vessels of the liver, andsecured in place by sutures. For parts or segments of liver, cannulaecan be placed in patent blood vessel openings on the cut surface, andsecured by sutures. In this case, small blood vessel openings usuallyneed to be sealed to prevent leakage of perfusion solutions from the cutsurface. The liver tissue is perfused with a divalent cation-free buffersolution preheated at 37° C. containing a cation-chelating agent, suchas, e.g., ethylenediamine tetraacetic acid (EDTA) or ethyleneglycoltetraacetic acid (EGTA). Buffer solutions can comprise salt solutions,such as, e.g., N-2-hydroxyethylpiperazine-N′-ethanesulfonic acid(HEPES), Williams E medium, Hank's balanced salt solution, or Earl'sbalanced salt solution, and can also include salts such as NaCl and KCl,among others. This leads to disruption of the desmosomal structures thathold cells together. The tissue is then perfused with the buffersolution containing divalent cation(s), such as Ca²⁺ and Mg²⁺, andmatrix-degrading enzymes that act to digest the tissue. The primaryliver cells, esp. hepatocytes, are usually released by gentle mechanicaldisruption, e.g., raking with a comb, shaking, pressing through filters,e.g., stainless steel filters, cheesecloth or nylon fabric, tomechanically complete the cell dissociation process. Such filters mayhave sieve sizes that allow passage of hepatocytes there through, bymeans of example and not limitation, about 0.1 mm or more, about 0.25 mmor more, about 0.50 mm or more, about 1 mm or more, or about 2 mm, 3 mm,4 mm or 5 mm. A succession of filters with progressively smaller sievesizes may be used to gradually disassociate the tissue and releasecells. The dissociated cells are rinsed with a buffer containingprotease inhibitor, serum and/or plasma to inactivate the collagenaseand other enzymes used in the perfusion process, separated by low speedcentrifugation, e.g., at between 10×g and 500×g (advantageously,substantially all live cells can be pelleted, while dead cells and celldebris are substantially eliminated), and the pellets obtained arewashed with ice-cold buffer solution to purify the cell suspension.

The number and quality of the isolated liver cells can vary depending,e.g., on the quality of the tissue used, the compositions of perfusionbuffer solutions, and the type and concentration of enzyme. Frequentlyused enzymes include, but are not limited to, collagenase, pronase,trypsin, dispase, hyaluronidase, thermolysin and pancreatin, andcombinations thereof. Collagenase is most commonly used, often preparedfrom bacteria (e.g. from Clostridium histolyticum), and may oftenconsist of a poorly purified blend of enzymes, which may haveinconsistent enzymatic action. Some of the enzymes exhibit proteaseactivity, which may cause unwanted reactions affecting the quality andquantity of viable/healthy cells. It is understood by those of skill inthe art to use enzymes of sufficient purity and quality to obtain viableliver cell populations.

Other methods of harvesting primary liver cells may exclude enzymaticdigestion techniques. Mechanical disruption has been widely used,although the yields of liver cells produced by this approach tend to beless than by collagenase digestion, as well as being less consistent.However, recent methods involving sucrose-EDTA perfusion in combinationwith controlled vibration in a cooled environment have been developedwith reasonable success (Kravchenko et al. 2002. Cell Biol Int 26:1003-1006). The liver perfusion is performed in situ using a solution ofsucrose containing EDTA (pH 7.4). After perfusion, the liver is removedfrom the body, placed into a dish, and divided finely in a small volumeof ice-cold medium. The cells of the liver fragments are liberated bymeans of controlled mechanical vibrational disaggregation (MVD), using ahomogenizer motor. The resultant slurry produced by this method can thenbe filtered through coarse mesh to give an initial suspension of livercells. The cells can be suspended in medium, and recovered bycentrifugation. Accordingly, in an embodiment, the disassociation ofliver or part thereof may be by mechanical disruption.

A skilled person is aware that two-step collagenase techniques may beparticularly suited to release at least hepatocytes from liver tissue.Cell suspensions obtained using the said technique may comprise aconsiderable proportion of hepatocytes, and may also contain other livercell types. As mentioned, the present inventors have realised that suchcell suspensions are a particularly suitable starting material forobtaining the progenitor or stem cell of the invention.

In an embodiment, the method of disassociating liver or part thereof mayform a cell suspension (as could be easily optimised by a skilledperson) comprising at least 10%, e.g., at least 20%; at least 30%, e.g.,at least 40%; at least 50%, e.g., at least 60%; at least 70%, e.g., atleast 80%; or at least 90%, or up to about 100% of individual cells,i.e., single cells.

As mentioned, disassociating liver tissue thus provides a population ofprimary cells from the said adult liver or part thereof.

As used herein, the term “primary cell” includes cells present in asuspension of cells obtained from a tissue or organ of a subject, e.g.,by disassociating thereof (i.e., a cell population prior to its beingplated), cells present in an explanted tissue, both of the previouskinds of cells when first time plated, and cells of the cell suspensionsderived from these first time plated cells. The term “secondary cell”refers to cells at all subsequent steps in cultivation. Hence, whenprimary cells plated for the first time are passaged, e.g., lifted froma substrate surface and re-plated, they are then referred to herein assecondary cells, as are all cells in subsequent passages.

The population of primary cells as defined and obtained herein bydisassociating liver or part thereof may typically be heterogeneous,i.e., it may comprise cells belonging to more than one cell type thatare comprised in liver. Exemplary liver-constituting cell types includebut are not limited to hepatocytes, cholangiocytes (bile duct cells),Kupffer cells, hepatic stellate cells (Ito cells), oval cells and liverendothelial cells. The above terms have art-established meanings and areconstrued broadly herein as encompassing any cell type classified assuch. Liver-constituting cell types further encompass both parenchymaland non-parenchymal liver cells.

By means of further illustration but not limitation, “hepatocyte”encompasses epithelial, parenchymal liver cells, including but notlimited to hepatocytes of different sizes (e.g., “small”, “medium-size”and “large” hepatocytes), ploidy (e.g., diploid, tetraploid, octaploid)or other characteristics. For example, some authors propose that “large”hepatocytes, as defined therein, are the parenchymal cells responsiblefor physiological functions of the liver, while “small” hepatocytesprovide a reservoir progenitor cells committed to development towardshepatocytes (see, e.g., Mitaka et al. Biochem Biophys Res Commun 214:310-7, 1995). Further by means of illustration and not limitation,“cholangiocytes” encompass epithelial cells of the bile ducts. Also bymeans of illustration and not limitation, “oval cell” encompass cells ofdistinctive morphology (e.g., nucleus shape) and cell marker expression,as known in the art, which are proposed to be progenitor cells capable,under certain conditions, of giving rise to hepatocytes and bile ductcells (Lowes et al. KN. 2003. J Gastroenterol Hepatol 18: 4-12; Yi et al1999. J of Hepatology 31: 497-507).

Hence, in an embodiment, a heterogeneous population of primary livercells may comprise cells of at least two, e.g., at least three or atleast four or more liver-constituting cell types, e.g., cells belongingto all or substantially all liver-constituting cell types, including butnot limited to the above listed liver cell types. A skilled person willappreciate that the heterogeneous population may comprise liver celltypes which have been previously described as such, whether in vivo orin vitro, as well as liver cell types which have not been previouslydescribed, classified, isolated and/or characterised in the art.

A skilled person will also appreciate that the heterogeneous cellpopulation may but need not comprise various liver cell types in thesame or substantially the same relative proportions as present in theliver or part thereof having been disassociated. For example, a skilledperson knows that particular manners of disassociating liver tissues maylead to more effective isolation of one or more cell types compared toone or more other cell types, whereby the obtained cell suspension maybe inadvertently or purposefully enriched for the former one or morecell types. Moreover, some disassociating methods may differently affectthe survival and/or viability of different liver cell types. Also, askilled person is well aware of art methods for enriching a cellpopulation obtained by disassociating liver or part thereof for one ormore desired liver cell types. Such methods include but are not limitedto differential centrifugation, buoyant density gradient centrifugation,filtration, cell elutriation, affinity purification, protease digestion,or the like.

In an embodiment, the heterogeneous population of primary liver cellsmay comprise cells belonging to all or substantially allliver-constituting cell types. The present inventors realised a methodfor obtaining a previously undisclosed type of progenitor or stem cellfrom liver. Nevertheless, the inventors wish not to be bound by anyhypothesis as to the origin of the said novel progenitor or stem cell.

By means of example and not limitation, the said progenitor or stemcell, or an ancestor thereof, may have been present in liver, e.g., inparenchyma or non-parenchyma thereof. For example, such ancestor mayhave had a phenotype identical to, similar to or different from theisolated progenitor or stem cell (e.g., culturing according to theinvention may have altered the phenotype of the ancestor).Alternatively, or in addition, the isolated progenitor or stem cell mayhave arisen due to alteration, e.g., differentiation ordedifferentiation, of one or more liver cell types, e.g., a liver celltype that has or has not been previously known. In view hereof, themethod of the present invention may preferably start from a cellpopulation representative of all or substantially all liver cell types.

The inventors have realised that the progenitor or stem cell of theinvention can be advantageously obtained from a cell population formedby disassociating liver or part thereof, wherein the said cellpopulation comprises hepatocytes. Hence, a suitable method fordisassociating liver or part thereof according to the invention forms acell population comprising hepatocytes. Without being bound to anytheory, the inventors think that the progenitor or stem cell of theinvention, or an ancestor thereof, is co-released from liver tissue bydisassociating the liver in a manner which releases at least hepatocytesfrom the liver.

In an embodiment, the method of disassociating liver or part thereof mayform a cell population comprising a proportion of hepatocytes which isat least about 10%, at least about 20%, at least 30%, at least 40%,preferably at least about 50%, e.g., at least 60%, more preferably atleast about 70%, e.g., at least about 80%, even more preferably at leastabout 90% or more, such as at least about 95%, at least about 96%, atleast about 97%, at least about 98%, or at least about 99%. Theinventors realised that method of disassociating liver or part thereofwhich forms a cell population comprising a substantial proportion ofhepatocytes, e.g., at least about 50% or more as above, providessuitable starting cell population for obtaining the progenitor or stemcell of the invention.

In a preferred embodiment, a cell population comprising the aboveproportions of hepatocytes may be obtained by disassociating liver orpart thereof, without including steps for further enriching the cellpopulation for hepatocytes and/or other cell types.

In another embodiment, a cell population comprising the aboveproportions of hepatocytes may be obtained by disassociating liver orpart thereof and one or more further steps for enriching the cellpopulation for hepatocytes and/or other cell types, esp. forhepatocytes. However, a skilled person will appreciate that while theprogenitor or stem cell of the invention, or ancestor thereof, can bereleased from liver under disassociation conditions suitable forreleasing hepatocytes therefrom, it may, but need not always, co-purifywith hepatocytes, or one or more sub-groups of hepatocytes (e.g.,“large” or “small” hepatocytes), or other cell types, in methods forenriching a cell population for hepatocytes, sub-groups thereof, orother cell types. It is within the ability of a skilled person to selectmethods for enriching hepatocytes or other cell types, esp. forhepatocytes, that retain the progenitor or stem cell of the invention,or ancestor thereof, in the resulting cell population.

Further, a skilled person will understand that the progenitor or stemcell of the invention, or an ancestor thereof, may have certainproperties (e.g., physical properties or surface marker expression)which may allow its enrichment in the cell population obtained fromliver, using a suitable separation technique. It may be within the reachof a skilled person to determine which fraction of a cell populationseparated on basis of one or more criteria comprises, or is enrichedfor, the progenitor or stem cell of the invention, or an ancestorthereof. This can be done, e.g., by cultivating cells from the varioustest fractions according to the method of the invention and ascertainingwhich fraction(s) yield the progenitor or stem cell of the invention.

An “enriched population” of cells refers to a population of cells inwhich one or more cell types are present in greater relative proportionsthan that which could be found in vivo or in the cell populationsubjected to enrichment.

Plating of Primary Cells from Liver Tissue

The method of the invention comprises culturing the primary cellpopulation obtained by dissociating liver tissue as explained. To thisaim, the primary population of liver cells is plated onto a substratewhich allows adherence of cells thereto.

The term “plating” as used herein is synonymous to seeding orinoculating, and in general refers to introducing a cell population intoan in vitro environment capable of promoting the survival and/or growthof the introduced cells. Typically, the said environment may be providedin a system which is suitably delimited from the surroundings, such thatit can prevent an undesired exchange of matter between the saidenvironment and the surroundings (thereby avoiding, e.g., contaminationof the environment or escape of culture medium or cells therefrom),while it can allow for continuous or intermittent exchange of other,useful, matter components between the said environment and thesurroundings (e.g., an occasional exchange of a part or all of theculture medium, the continuous exchange of gases, or the harvesting ofthe cells after culturing, etc.). Usually, environments suitable forculturing of cell can be generated in culture vessels well-known in theart, such as, e.g., cell culture flasks, well plates and dishes ofvarious formats.

In the present invention, cells (e.g., primary liver cells) are platedonto a substrate which allows for adherence of cells thereto, i.e., asurface which is not generally repulsive to cell adhesion or attachment.This may be carried out, e.g., by plating the cells in a culture system(e.g., a culture vessel) which displays one or more substrate surfacescompatible with cell adhesion. When the said one or more substratesurfaces contact the suspension of cells (e.g., suspension in a medium)introduced into the culture system, cell adhesion between the cells andthe substrate surfaces may ensue. Accordingly, the term “plating onto asubstrate which allows adherence of cells thereto” refers to introducingcells into a culture system which features at least one substratesurface that is generally compatible with adherence of cells thereto,such that the plated cells can contact the said substrate surface.General principles of maintaining adherent cell cultures are well-knownin the art.

In general, a substrate which allows adherence of cells thereto may beany substantially hydrophilic substrate. As known in the art, culturevessels, e.g., culture flasks, well plates, dishes, or the like, may beusually made of a large variety of polymeric materials, including, butnot limited to polyacrylates, polymethylacrylates, polycarbonates,polystyrenes, polysulphones, polyhydroxyacids, polyanhydrides,polyorthoesters, polyphosphazenes, polyphosphates, polyesters, nylons ormixtures thereof, etc. Generally, culture vessels made of such materialsare surface treated after moulding in order to provide for hydrophilicsubstrate surfaces and thereby enhance the likelihood of effective cellattachment. Surface treatment may take the form of a surface coating, ormay involve the use of directed energy at the surface with the intentionof generating chemical groups on the polymer surface. These chemicalgroups will have a general affinity for water or otherwise exhibitsufficient polarity to permit stable adsorption to another polar group.These functional groups lead to hydrophilicity and or an increase insurface oxygen and are properties recognized to enhance cell growth onso modified substrate surfaces. Such chemical groups may include groupssuch as amines, amides, carbonyls, carboxylates, esters, hydroxyls,sulfhydryls and the like. Examples of directed energy includeatmospheric corona discharge, radio frequency (RF) vacuum plasmatreatment, and DC glow discharge or plasma treatment (e.g., U.S. Pat.No. 6,617,152). Current standard practices for growing adherent cellsmay involve the use of defined chemical media with addition of bovine,human or other animal serum. The added serum, besides providingnutrients and/or growth promoters, may also promote cell adhesion bycoating the treated plastic surfaces with a layer of matrix to whichcells can better adhere.

An alternative substrate surface compatible with cell adhesion may beglass, optionally surface treated to introduce functional groups, e.g.,as listed above, to increase the hydrophilicity thereof.

Other adherent substrate surfaces may be generated via surface coating,e.g., coating of the polymeric or treated polymeric surfaces as above.In a non-limiting example, the coating may involve suitablepoly-cations, such as, e.g., poly-ornithine or poly-lysine.

In another example, preferred coating, and accordingly the substrate,comprises one or more components of extracellular matrix, e.g., the ECMproteins fibrin, laminin, collagen, preferably collagen type 1,glycosaminoglycans, e.g., heparin or heparan sulphate, fibronectin,gelatine, vitronectin, elastin, tenascin, aggrecan, agrin, bonesialoprotein, cartilage matrix protein, fibrinogen, fibulin, mucins,entactin, osteopontin, plasminogen, restrictin, serglycin,SPARC/osteonectin, versican, thrombospondin 1, or cell adhesionmolecules including cadherins, connexins, selectins, by themselves or invarious combinations.

Preferred examples may include fibrin, laminin or collagen. Furtherpreferred examples may involve compositions comprising ECM components,such as, e.g., Matrigel® Basement Membrane Matrix (BD Biosciences),which is solubilised basement membrane preparation extracted from EHSmouse sarcoma, a tumour rich in ECM proteins, with laminin as a majorcomponent, followed by collagen type 4, heparan sulphate proteoglycans,and entactin.

A particularly preferred embodiment includes coating consisting of orcomprising collagen, esp. collagen type 1.

As appreciated by those skilled in the art, the cells may be counted inorder to facilitate subsequent plating of the cells at a desireddensity. Where, as in the present invention, the cells after plating mayprimarily adhere to a substrate surface present in the culture system(e.g., in a culture vessel), the plating density may be expressed asnumber of cells plated per mm² or cm² of the said substrate surface. Inthe present invention, the plating density of the primary cells obtainedfrom disassociating liver or part thereof may be between 1 cell/mm² and1×10⁶ cells/mm², e.g., between 1×10¹ and 1×10⁵ cells/mm² or between1×10² and 1×10⁵ cells/mm², e.g., between 1×10³ and 1×10⁵ cells/mm²,between 5×10³ and 5×10⁴ cells/mm², between 1×10¹ and 1×10³ cells/mm²,between 1×10² and 1×10⁴ cells/mm², e.g., about 1×10¹, 5×10¹, 1×10²,5×10², 1×10³, 5×10³, 6×10³, 7×10³, 8×10³, 9×10³, 1×10⁴, 2×10⁴, 3×10⁴,4×10⁴, 5×10⁴, or 1×10⁵, cells/mm².

Typically, after plating of the primary liver cells, the cell suspensionis left in contact with the adherent surface to allow for adherence ofcells from the cell population to the said substrate. In contacting theprimary liver cells with adherent substrate, the cells may beadvantageously suspended in an environment comprising at least a medium,in the methods of the invention typically a liquid medium, whichsupports the survival and/or growth of the cells. The medium may beadded to the system before, together with or after the introduction ofthe cells thereto. The medium may be fresh, i.e., not previously usedfor culturing of cells, or may comprise at least a portion which hasbeen conditioned by prior culturing of cells therein, e.g., culturing ofthe cells which are being plated or antecedents thereof, or culturing ofcells more distantly related to or unrelated to the cells being plated.

To facilitate said adherence, in embodiments, the primary cellsuspension may be contacted with the adherent surface for at least about0.5 h, e.g., for at least about 1 h, preferably for at least about 2 h,e.g., for at least about 4 h, more preferably for at least about 8 h,e.g., for at least about 12 h, even more preferably for at least about16 h, e.g., for at least about 20 h, and most preferably for at leastabout 24 h or more, e.g., for at least about 28, 32, 36, 40, 44 or 48 h.

In other preferred embodiments, the primary cell suspension may becontacted with the adherent surface for between about 2 h and about 48h, e.g., for between about 12 h and about 48 h, preferably for betweenabout 12 h and about 36 h, e.g., for between about 16 h and about 32 h,even more preferably for between about 20 h and about 28 h, and mostpreferably for about 24 h.

While the above times are preferred, shorter or longer may also providefor attachment of cells compatible with the present invention, and askilled person can optimise such times.

After cells from the primary liver cell population are allowed to attachto adherent substrate as described above, non-adherent matter is removedfrom the culture system. Non-adherent matter may comprise, but is notlimited to, cells that have not attached to the adherent substrate (suchas, e.g., cells which are not prone to adherence, or cells which wouldnot attach within the time allowed therefore), non-viable or dead cells,cell debris, etc. Non-adherent matter can be typically removed bydiscarding medium from the culture system, whereupon adherent cellsremain attached to the substrate, and optionally washing, once orrepeatedly, the adherent cells and the culture system with suitablemedium or isotonic buffer (e.g., PBS). Hereby, cells from the primaryliver cell population, which have adhered to the substrate surface, areselected for further culturing.

The environment in which the cells are plated and allowed to attach maycomprise at least a medium, in the methods of the invention typically aliquid medium, which supports the survival and/or growth of the cells.The medium may be added to the system before, together with or after theintroduction of the cells thereto. The medium may be fresh, i.e., notpreviously used for culturing of cells, or may comprise at least aportion which has been conditioned by prior culturing of cells therein,e.g., culturing of the cells which are being plated or antecedentsthereof, or culturing of cells more distantly related to or unrelated tothe cells being plated.

The medium may be a suitable culture medium as described elsewhere inthis specification. Preferably, the composition of the medium may havethe same features, may be the same or substantially the same as thecomposition of medium used in the ensuing steps of culturing theattached cells. Otherwise, the medium may be different. Advantageously,the medium can comprise serum or plasma, which may further facilitatecell adherence.

Culturing the Primary Cells from Liver Tissue

Cells from the primary cell population, which have adhered to the saidsubstrate, preferably in the said environment, are subsequently culturedfor at least 7 days, e.g., for at least 8 days or for at least 9 days,preferably for at least 10 days, e.g., at least 11 or at least 12 days,at least 13 days or at least 14 days, more preferably for at least 15days, e.g., for at least 16 days or for at least 17 days, or even for atleast 18 days, e.g., for at least 19 days or at least 20 days or more.The term “culturing” is common in the art and broadly refers tomaintenance and/or growth of cells and/or progeny thereof.

In embodiments, the primary cells may be cultured for at least betweenabout 10 days and about 40 days, preferably for at least between about15 days and about 35 days, e.g., for at least between about 15 days and20 days, such as for at least about 15, 16, 17, 18, 19 or 20 days.Preferably, the primary cells may be so cultured for no longer than 60days, or no longer than 50 days, or no longer than 45 days.

As appreciated by those skilled in the art, prolonged culturing of cellsin a culture system may necessitate regular exchange of the culturemedium for a fresh medium. A skilled person is capable of assessing theneed for exchanging the medium by inspecting the cell cultureparameters, such as, e.g., the pH thereof, the cell density or cellappearance. Typically, the medium may be changed at regular intervals,e.g., every 1 to 10 days, preferably between 16 and 32 hours (e.g.,about 24 hours) after plating and then preferably every 2 to 6 days, ormore preferably every 2 to 4 days, e.g., about every 2, 3 or 4 days. Thewhole volume of the medium may be changed or, alternatively, only partof the medium may be changed, such that a portion of the mediumconditioned by the previous culturing of the cells is retained. In anembodiment, substantially the whole volume of medium is exchanged forfresh medium. In another preferred embodiment, the medium is notexchanged during prolonged culturing of the cells.

The primary cell suspension and the further adherent cells are culturedin the presence of a liquid culture medium. Typically, the medium willcomprise a basal medium formulation as known in the art. Many basalmedia formulations (available, e.g., from the American Type CultureCollection, ATCC; or from Invitrogen, Carlsbad, Calif.) can be used toculture the primary cells herein, including but not limited to Eagle'sMinimum Essential Medium (MEM), Dulbecco's Modified Eagle's Medium(DMEM), alpha modified Minimum Essential Medium (alpha-MEM), BasalMedium Essential (BME), Iscove's Modified Dulbecco's Medium (IMDM), BGJbmedium, F-12 Nutrient Mixture (Ham), Leibovitz L-15, DMEM/F-12,Essential Modified Eagle's Medium (EMEM), RPMI-1640, Medium 199,Waymouth's MB 752/1 or Williams Medium E, and modifications and/orcombinations thereof. Compositions of the above basal media aregenerally known in the art and it is within the skill of one in the artto modify or modulate concentrations of media and/or media supplementsas necessary for the cells cultured. In an embodiment, a preferred basalmedium formulation may be Williams Medium E, which is a rich formulationreported to sustain in vitro culture of adult liver cells. Otherembodiments may employ further basal media formulations, such as chosenfrom the ones above.

Such basal media formulations contain ingredients necessary for mammalcell development, which are known per se. By means of illustration andnot limitation, these ingredients may include inorganic salts (inparticular salts containing Na, K, Mg, Ca, Cl, P and possibly Cu, Fe, Seand Zn), physiological buffers (e.g., HEPES, bicarbonate), nucleotides,nucleosides and/or nucleic acid bases, ribose, deoxyribose, amino acids,vitamins, antioxidants (e.g., glutathione) and sources of carbon (e.g.glucose, pyruvate, e.g., sodium pyruvate, acetate, e.g., sodiumacetate), etc. It will also be apparent that many media are available aslow-glucose formulations with or without sodium pyruvate.

For use in culture, basal media can be supplied with one or more furthercomponents. For example, additional supplements can be used to supplythe cells with the necessary trace elements and substances for optimalgrowth and expansion. Such supplements include insulin, transferrin,selenium salts, and combinations thereof. These components can beincluded in a salt solution such as, but not limited to, Hanks' BalancedSalt Solution (HBSS), Earle's Salt Solution. Further antioxidantsupplements may be added, e.g., β-mercaptoethanol. While many basalmedia already contain amino acids, some amino acids may be supplementedlater, e.g., L-glutamine, which is known to be less stable when insolution. A medium may be further supplied with antibiotic and/orantimycotic compounds, such as, typically, mixtures of penicillin andstreptomycin, and/or other compounds, exemplified but not limited to,amphotericin, ampicillin, gentamicin, bleomycin, hygromycin, kanamycin,mitomycin, mycophenolic acid, nalidixic acid, neomycin, nystatin,paromomycin, polymyxin, puromycin, rifampicin, spectinomycin,tetracycline, tylosin, and zeocin.

Hormones can also be advantageously used in cell culture and include,but are not limited to D-aldosterone, diethylstilbestrol (DES),dexamethasone, estradiol, hydrocortisone, insulin, prolactin,progesterone, somatostatin/human growth hormone (HGH), thyrotropin,thyroxine, L-thyronine, epithelial growth factor (EGF) and hepatocytegrowth factor (HGF). Liver cells can also benefit from culturing withtriiodithyronine, α-tocopherol acetate, and glucagon.

Lipids and lipid carriers can also be used to supplement cell culturemedia. Such lipids and carriers can include, but are not limited tocyclodextrin, cholesterol, linoleic acid conjugated to albumin, linoleicacid and oleic acid conjugated to albumin, unconjugated linoleic acid,linoleic-oleic-arachidonic acid conjugated to albumin, oleic acidunconjugated and conjugated to albumin, among others. Albumin cansimilarly be used in fatty-acid free formulations.

Also contemplated is supplementation of cell culture medium withmammalian plasma or sera. Plasma or sera often contain cellular factorsand components that are necessary for viability and expansion. The useof suitable serum replacements is also contemplated.

The term “plasma” is as conventionally defined. Plasma is usuallyobtained from a sample of whole blood, which is provided or contactedwith an anticoagulant, such as heparin, citrate (e.g., sodium citrate oracid citrate dextrose), oxalate or EDTA, upon or shortly after drawingthe blood sample, to prevent clotting. Subsequently, cellular componentsof the blood sample are separated from the liquid component (plasma) byan appropriate technique, typically by centrifugation. The term “plasma”therefore refers to a composition which does not form part of a human oranimal body.

The term “serum” is as conventionally defined. Serum can be usuallyobtained from a sample of whole blood by first allowing clotting to takeplace in the sample and subsequently separating the so formed clot andcellular components of the blood sample from the liquid component(serum) by an appropriate technique, typically by centrifugation. Aninert catalyst, e.g., glass beads or powder, can facilitate clotting.Advantageously, serum can be prepared using serum-separating vessels(SST) known in the art, which contain the inert catalyst to facilitateclotting and further include a gel with density designed to becomepositioned between the liquid component and the clot and cellularcomponents after centrifugation, thus simplifying separation.Alternatively, serum can be obtained from plasma by removing theanticoagulant and fibrin. The term “serum” hence refers to a compositionwhich does not form part of a human or animal body.

The isolated plasma or serum can be used directly in the methods of thepresent invention. They can also be appropriately stored for a later usein the method of the present invention. Typically, plasma or serum canbe stored for shorter time periods, e.g., up to about 1-2 weeks, at atemperature above the respective freezing points of plasma or serum, butbelow ambient temperature. Usually, this temperature will be about 15°C. or less, preferably about 10° C. or less, more preferably about 5° C.or less, e.g., about 5° C., 4° C., 3° C., 2° C. or about 1° C., mostpreferably about 5° C. or about 4° C. Alternatively, plasma or serum canbe stored at below their respective freezing points, i.e., by freezestorage. As usual in the art, advantageous temperatures for freezestorage of plasma or serum can be about −70° C. or less, e.g., about−75° C. less or about −80° C. or less. Such temperatures mayadvantageously prevent any thawing of the stored plasma or serum,thereby preserving the quality thereof. Freeze storage can be usedirrespective of the time period for which the plasma or serum need to bestored, but may be particularly suitable if longer storage is required,e.g., for longer than a few days or for longer than 1-2 weeks.

Prior to storage or use, the isolated plasma or serum can be heatinactivated. Heat inactivation is used in the art mainly to remove thecomplement. Heat inactivation typically involves incubating the plasmaor serum at 56° C. for 30 to 60 min, e.g., 30 min, with steady mixing,after which the plasma or serum is allowed to gradually cool to ambienttemperature. A skilled person will be aware of any common modificationsand requirements of the above procedure.

Optionally, the plasma or serum may also be sterilised prior to storageor use. Usual means of sterilisation may involve, e.g., filtrationthrough one or more filters with pore size smaller than 1 μm, preferablysmaller than 0.5 μm, e.g., smaller than 0.45 μm, 0.40 μm, 0.35 μm, 0.30μm or 0.25 μm, more preferably 0.2 μm or smaller, e.g., 0.15 μm orsmaller, 0.10 μm or smaller.

Suitable sera or plasmas for use in the media of the present inventionmay include human serum or plasma, or serum or plasma from non-humananimals, preferably non-human mammals, such as, e.g., non-human primates(e.g., lemurs, monkeys, apes), foetal or adult bovine, horse, porcine,lamb, goat, dog, rabbit, mouse or rat serum or plasma, etc. In anotherembodiment, the invention foresees the use of any combination of theabove plasmas and/or sera.

Accordingly, in an embodiment, the serum or plasma may be obtained froman organism of the same species as is the species from which the primaryliver cells are obtained. In a non-limiting example, human serum orplasma may be used for culturing primary human liver cells.

In another preferred embodiment, the medium comprises bovine serum orplasma, preferably foetal bovine (calf) serum or plasma, more preferablyfoetal bovine (calf) serum (FCS or FBS).

In another embodiment, the medium used to culture primary human livercells comprises bovine serum or plasma, preferably foetal bovine (calf)serum or plasma, more preferably foetal bovine (calf) serum (FCS orFBS).

In embodiments, the medium comprises between about 0.5% and about 40%(v/v) of serum or plasma or serum replacement, preferably between about5% and 20% (v/v), e.g., between about 5% and 15% (v/v), more preferablybetween about 8% (v/v) and about 12% (v/v), e.g., about 10% (v/v) ofserum or plasma or serum replacement, esp. the preferred serum or plasmaas defined above.

In a further preferred embodiment, the medium used to culture primaryhuman liver cells comprises bovine serum or plasma, preferably foetalbovine (calf) serum or plasma, more preferably foetal bovine (calf)serum (FCS or FBS), in the amount of between about 0.5% and about 40%(v/v), preferably between about 5% and 20% (v/v), e.g., between about 5%and 15% (v/v), more preferably between about 8% (v/v) and about 12%(v/v), e.g., about 10%.

In yet other embodiments, a medium may comprise plasma or serum derivedfrom more than one species. For example, the medium may comprise amixture of serum or plasma derived from a species corresponding to thecultured primary liver cells, and from another species. For example, amedium for culturing human liver cells may comprise a mixture of humanplasma or serum, preferably human serum, and bovine plasma or serum,preferably bovine serum.

Further, the medium may preferably comprise at least one exogenously(i.e., in addition to the plasma or serum) added growth factor. Askilled person would appreciate that the ordinary components of basalmedia (before addition of serum or plasma), e.g., in particular,isotonic saline, buffers, inorganic salts, amino acids, carbon sources,vitamins, antioxidants, pH indicators and antibiotics, are notconsidered growth factors or differentiation factors in the art. On theother hand, serum or plasma is a complex composition possibly comprisingone or more such growth factors or differentiation factors.

The term “growth factor” as used herein refers to a biologically activesubstance which influences proliferation, growth, differentiation,survival and/or migration of various cell types, and may effectdevelopmental, morphological and functional changes in an organism,either alone or when modulated by other substances. A growth factor maytypically act by binding, as a ligand, to a receptor (e.g., surface orintracellular receptor) present in cells responsive to the growthfactor. A growth factor herein may be particularly a proteinaceousentity comprising one or more polypeptide chains.

By means of example and not limitation, the term “growth factor”encompasses the members of the fibroblast growth factor (FGF) family,bone morphogenic protein (BMP) family, platelet derived growth factor(PDGF) family, transforming growth factor beta (TGF-beta) family, nervegrowth factor (NGF) family, the epidermal growth factor (EGF) family,the insulin related growth factor (IGF) family, the hepatocyte growthfactor (HGF) family, hematopoietic growth factors (HeGFs), theplatelet-derived endothelial cell growth factor (PD-ECGF), angiopoietin,vascular endothelial growth factor (VEGF) family, glucocorticoids, andthe like.

In a preferred embodiment, the medium comprises a growth factor which isa member of the epidermal growth factor (EGF) family. In a furtherembodiment, the said member of the EGF family is any chosen from thegroup consisting of amphiregulin, betacellulin, EGF, epiregulin, HB-EGF(heparin-binding EGF-like growth factor), NRG1 (neuregulin-1) isoformGGF2, NRG1 isoform SMDF, NRG1-alpha, NRG1-beta, TGFalpha, Tomoregulin-1and TMEFF2. In a particularly preferred embodiment, the medium comprisesEGF.

In a further embodiment, the medium comprises a growth factor which is amember of the insulin related growth factor (IGF) family. In a furtherembodiment, the said member of the IGF family is any chosen from thegroup consisting of insulin, IGF1A (insulin-like growth factor 1A),IGF1B, IGF2, INSL3 (insulin-like 3), INSL5, INSL6 and relaxin. In aparticularly preferred embodiment, the medium comprises insulin.

In a further embodiment, the medium comprises a growth factor which is aglucocorticoid. In a further embodiment, the said glucocorticoid is anychosen from the group consisting of dexamethasone, hydrocortisone,prednisolone, methylprednisolone, prednisone, triamcinolone,corticosterone, fluocinolone, cortisone, betamethasone. In aparticularly preferred embodiment, the medium comprises dexamethasone.

In further preferred embodiments, the medium may comprise a combinationof any two or more exogenously added growth factors or preferred growthfactors as defined above. By means of example and not limitation, themedium may comprise EGF and insulin, or EGF and dexamethasone, orinsulin and dexamethasone, or each EGF, insulin and dexamethasone; amedium comprising these exogenously added growth factors may preferablycomprise serum or plasma as defined in the above embodiments.

A skilled person is in general knowledgeable of concentrations in whichparticular growth factors can induce an effect, esp. on in vitrocultured cells, and can use such concentrations for the above recitedgrowth factors. By means of example and not limitation, EGF may betypically used at concentrations between about 0.1 ng/ml and 1 μm/ml andpreferably between 1 ng/ml and 100 ng/ml, e.g., at about 25 ng/ml;insulin can be typically used at concentrations between about 0.1 μm/mland 1 mg/ml and preferably between about 1 μm/ml and 100 μm/ml, e.g., atabout 10 μm/ml; dexamethasone can be typically used at concentrationsbetween about 0.1 nM and 1 μM, preferably between about 1 nM and 100 nM,e.g., at about 10 nM.

In a preferred embodiment, esp. where the method is used for human livercells, the growth factor used in the present method may be a humangrowth factor. As used herein, the term “human growth factor” refers toa growth factor substantially the same as a naturally occurring humangrowth factor. For example, where the growth factor is a proteinaceousentity, the constituent peptide(s) or polypeptide(s) thereof may haveprimary amino acid sequence identical to a naturally occurring humangrowth factor. The use of human growth factors in the present method ispreferred, as such growth factors are expected to elicit a desirableeffect on cellular function.

As described, the present inventors have realised that by culturingprimary liver cells for time durations as defined above, and preferablyusing media compositions as described above, a progenitor or stem cellof the invention emerges and proliferates, while differentiated livercell types become less prevalent in the prolonged culturing of theprimary liver cells. Without being limited to any hypothesis, thedifferentiated liver cell types may, for example, fail to proliferate,die and/or retro-differentiate during the prolonged culturing. Asdetailed in the experimental section, the progenitor or stem cell may bedistinguished from other cell types present in the primary cell cultureby, among others, its morphology, which according to the inventors'knowledge may be denoted as mesenchymal or mesenchymal-like morphologyand may typically comprise a flattened form, broad cytoplasm and ovoidnuclei with one or two nucleoli.

The inventors also realised that the emergence, proliferation andenrichment of the primary culture for the said progenitor or stem cellmay be further promoted by altering the culture medium such that thisfavours further elimination of one or more differentiated liver celltypes, esp. of hepatocytes which may prevail in a culture of isolatedprimary liver cells. In such conditions, the progenitor or stem cell ofthe invention can advantageously proliferate and become a prevalent celltype in the primary liver cell culture. The differentiated liver celltypes can be lost, e.g., because one or more differentiated cell typesare physically decreased or eliminated during culture; alternatively,the differentiated phenotypes can be decreased because one or more celltypes retro-differentiates during culture.

Any medium which favours the elimination of one or more differentiatedliver cell types, esp. hepatocytes, while sustaining the proliferationof the progenitor or stem cell of the invention (as can be readilyjudged, for example, by visual inspection of the cell culture for theprevalence of the different cell types) can be used. By means ofillustration, as exemplified by the inventors, one alteration may be theuse of a basal medium comprising high glucose concentration, e.g., aconcentration between 3000 mg/l and 6000 mg/l, preferably between 4000mg/l and 5000 mg/l, and typically about 4500 mg/l. A further alterationmay be the absence of exogenously added (i.e., in addition to thosepresent in the serum or plasma) growth factors. By means of an example,at least exogenously added insulin, dexamethasone and/or EGF may beabsent from the medium. Yet a further alteration may be the use of basalmedium other than Williams Medium E (which may be particularly suitedfor long-term culture of primary liver cell types, esp. hepatocytes). Bymeans of example and not limitation, basal media such as MEM, DMEM,alpha-MEM or EMEM may be advantageously used. It is to be understoodthat the medium may be altered in one, more than one or all of the aboveways. It is further to be understood that the medium comprises serum orplasma or serum replacement as described above, including the abovedetailed preferred embodiments thereof.

In an embodiment, a medium favouring elimination of one or moredifferentiated liver cell types and promoting the progenitor or stemcell of the invention as described above may be added at the outset ofculturing of the primary liver cells. In another embodiment, the mediummay be so altered during the prolonged culturing of the primary livercells. By means of example, the medium may be so altered starting atabout 1 day, at about 2 days, 3 days, e.g., at about 4 or 5 days, orstarting at about 6 days, such as, e.g., at about 7 or 8 days, orstarting at about 9 days, such as e.g., at about 10 or 11 days, startingat about 12 days, e.g., at about 13 or 14 days, more preferably startingat about 15 days, e.g., at about 16, 17, 18, 19 or 20 days followingplating of the primary liver cells. In an exemplary embodiment, themedium may be so altered at between 16 and 32 hours following plating,e.g., at about 24 hours. In embodiments, after the medium is so altered,it can be exchanged/refreshed, e.g., every 2 to 6 days, preferably every2 to 4 days, e.g., every 3 or 4 days.

Hence, in an exemplary preferred embodiment, primary liver cells may becultured following plating in a medium favouring survival of primaryliver cell types, including differentiated liver cell types, such ashepatocytes, and at the above time the medium may be altered to a mediumfavouring elimination of one or more differentiated liver cell types,incl. hepatocytes, and promoting the progenitor or stem cell of theinvention. For example, primary liver cells may first be cultured in amedium having one, more than one or all of the following features:comprising a rich basal medium, such as, e.g., Williams Medium E,comprising low glucose, e.g., between 500 mg/l and 2999 mg/l, andpreferably between 1000 mg/l and 2000 mg/l, comprising at least oneexogenously added growth factor and preferably one, more than one or allof insulin, dexamethasone and EGF. Thereafter, at the above times, themedium may be altered to include one, more than one or all of thefollowing features: comprising basal medium other than Williams MediumE, e.g., basal media such as MEM, DMEM, alpha-MEM or EMEM, comprisinghigh glucose, not comprising exogenously added growth factors or notcomprising at least dexamethasone, insulin and/or EGF. It is to beunderstood that the above media would comprise serum or plasma or serumreplacement as described above, including the above detailed preferredembodiments thereof.

As described, the above culturing of primary liver cells leads toemergence and proliferation of a progenitor or stem cell of theinvention in the culture. The said culturing can be advantageouslycontinued until the emerging progenitor or stem cell of the inventionhave proliferated sufficiently. For example, the said culturing can becontinued until the cell population achieved a certain degree ofconfluence, e.g., at least 40%, preferably at least 50%, more preferablyat least 60% and even more preferably at least 70%, e.g., at least 80%or at least 90% or more confluence. The term “confluence” as used hereinrefers to a density of cultured cells in which the cells contact oneanother covering substantially all of the surfaces available for growth(i.e., fully confluent).

Passaging of the Cells

Following the above culturing of the primary liver cells and emergenceand proliferation of the progenitor or stem cells of the invention, thecell population so obtained may be passaged at least once.

The present inventors surprisingly realised that the progenitor or stemcells of the invention that emerged in the primary cell culturesubstantially retain their proliferation capacity after passaging, whichthus allows to advantageously further enrich the cell population forthese cells. For sake of simplicity, the passage performed at this stageof the method is herein referred to as “first passage” (or passage 1)within the method of the invention. The cells may be passaged at leastone time and preferably two or more times. Each passage subsequent topassage 1 is referred to herein with a number increasing by 1, e.g.,passage 2, 3, 4, 5, etc.

When passaged, the cultured cells are detached and dissociated from theculture substrate and from each other. Detachment and dissociation ofthe cells can be carried out as generally known in the art, e.g., byenzymatic treatment with proteolytic enzymes (e.g., chosen from trypsin,collagenase, e.g., type I, II, III or IV, dispase, pronase, papain,etc.), treatment with bivalent ion chelators (e.g., EDTA or EGTA) ormechanical treatment (e.g., repeated pipetting through a small borepipette or pipette tip), or any combination of these treatments.Preferably, the detachment and dissociation of the cultured cells wouldyield a substantial proportion of the cells as single cells. Forexample, 40% or more of the cells can be recovered as single cells,e.g., at least 50%, preferably at least 60%, e.g., at least 70%, morepreferably at least 80%, e.g., at least 90% or at least 95% of the cellsmay be recovered as single cells. Moreover, the remaining cells may bepresent in cell clumps or clusters the majority of which can contain arelatively small number of cells, e.g., on average, between more than 1and 10 cells, e.g., less than 8 cells, preferably less than 6 cells,more preferably less than 4 cells, e.g., less than 3 or less than 2cells.

Typically, a suitable method of cell detachment and dispersion shouldpreserve viability of the cells. Preferably, a cell suspension obtainedfollowing detachment and dispersion may comprise at least 60% of viablecells, e.g., at least 70%, more preferably at least 80%, and mostpreferably at least 90% and up to 100% of viable cells. A skilled personwill know in general to choose conditions which ensure a desired degreeof cell detachment and dispersion, while preserving cell viability.

Next, the so detached and dissociated cells (typically as a cellsuspension in an isotonic buffer or a medium) are re-plated onto asubstrate which allows for adherence of cells thereto, and aresubsequently cultured in a medium as described above sustaining thefurther proliferation the progenitor or stem cells of the invention.Typically, the cells may be re-plated at plating density of between1×10¹ and 1×10⁶ cells/cm², e.g., between 1×10² and 1×10⁶ cells/cm², andpreferably between 1×10³ and 1×10⁵ cells/cm², e.g., at about 1×10³cells/cm², at about 5×10³ cells/cm², at about 1×10⁴ cells/cm², at about5×10⁴ cells/cm², or at about 1×10⁵ cells/cm2, and preferably at betweenabout 1×10³ and 1×10⁴ cells/mm².

Alternatively, the cells may be re-plated at a splitting ratio of, e.g.,between about ⅛ and ½, preferably between about ¼ and ½, and morepreferably at about ½ or about ⅓. The splitting ratio denotes thefraction of the passaged cells which is seeded into an empty (typicallya new) culture vessel of the same surface area as the vessel from whichthe cells were obtained.

The adherent substrate onto which the cells are re-plated is asdescribed in detail elsewhere in this specification. The substrate maypreferably be of the same kind as the substrate onto which the primaryliver cells were plated, including preferred embodiments of suchsubstrate described above, or may be different. Preferably, thissubstrate is also collagen, esp. collagen type I as described above.

The so passaged cells are further cultured, advantageously until thecells have become at least 50% confluent, e.g., at least 60%, preferablyat least 70%, e.g., at least 80%, more preferably at least 90%, e.g., atleast 95% or even fully confluent.

The present inventors have realised that while the cell populationobtained at this stage of the method comprises a substantial fraction ofthe progenitor or stem cells of the invention, it can be advantageouslypassaged at least one more time (i.e., at least a second passage),essentially as described above for the first passage. This furtherincreases the proportion of progenitor or stem cells of the invention inthe cell population, as judged by morphology and/or marker analysis, andeven a substantially homogeneous population of the progenitor or stemcells of the invention may be obtained.

Hence, according to the invention, following the plating and culture ofprimary liver cells leading to emergence and proliferation of theprogenitor or stem cells in the said culture, the cultured cells arepassaged at least once (i.e., first passage) and preferably at least twotimes (i.e., first and second passage), and optionally more times (i.e.,first, second and each subsequent passage). For example, the cells maybe passaged at least once, at least 2 times, at least 3 times, at least4 times or at least 5 times following the plating of primary livercells. In another embodiment, the cells may be passaged between 2 and 10times, e.g., between 2 and 8 times, or between 2 and 5 times, followingthe plating of primary liver cells. The additional passages (e.g., celldetachment and dispersion, replating, substrate, etc.) and culturing(e.g., medium, medium changes, resulting confluence, etc.) may beperformed at conditions substantially identical or analogous to those ofthe first passage, as described above, including preferred embodimentsthereof and may include modifications which would be obvious to theskilled person.

The method of the invention may thus provide for a cell populationcomprising a considerable fraction of progenitor or stem cells, asdefined in this disclosure, and the fraction of the said progenitor orstem cells can be increased by one or more passages of the prolongedculture of the primary liver cells. Typically, the population willcomprise at least about 10%, e.g., at least about 20% of the saidprogenitor or stem cells, but the inventors found that typically higherproportions of the said progenitor or stem cells will be obtained, e.g.,at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, or at least at least 90% or more. Moreover, the method mayeven yield a substantially homogeneous or homogeneous population of thesaid progenitor or stem cells. The fraction of the progenitor or stemcells can be evaluated by any appropriate standard method, e.g., by flowcytometry.

Maintaining the Obtained Cells

When a population comprising the liver-derived progenitor or stem cellsof the invention is achieved by the methods of the invention, andpossibly further enriched for the said progenitor or stem cells, thecell population may next be maintained and/or propagated in conditionsthat allow for growth and doubling of the said progenitor or stem cellswithout differentiation. Such conditions may be, e.g., the ones used forobtaining the progenitor or stem cells. A skilled person who is capableof assessing the presence or absence of cell differentiation may readilyestablish further conditions. This can advantageously increase thenumber of the progenitor or stem cells available for further usethereof.

The present inventors have realised that the primary or any othersubsequent passage could be cryopreserved for further use, as generallyknown in the art for mammalian cells.

The present inventors have realised that the progenitor or stem cells ofthe invention that emerged in the primary cell culture substantiallyretain their proliferation capacity after freezing and thawing. The saidcells may be stored as a frozen concentrated cell suspension, thawed, asgenerally done in the art, and re-plated in the same conditions asdescribed elsewhere in this specification.

Progenitor or Stem Cells of the Invention

The present inventors realised that the cell population obtained uponculture and preferably also passaging of primary liver cells asdescribed above comprises liver-derived progenitor or stem cells of theinvention, which co-express (i.e., are positive for) at least onemesenchymal marker, esp. one, more than one, e.g., 2, 3 or 4, or all ofthe markers CD90, CD73, CD44, vimentin and α-smooth muscle actin (ASMA),with the hepatocyte marker albumin (ALB) and possibly with one or moreother hepatic or hepatocyte markers, preferably one, more than one, orall CD29, alpha-fetoprotein (AFP), alpha-1 antitrypsin and/or MRP2transporter.

In a more particular embodiment, the liver-derived progenitor or stemcells of the invention may co-express (i.e., are positive for) at leastone mesenchymal marker, esp. one, more than one, e.g., 2, 3 or 4, or allof the markers CD90, CD73, CD44, vimentin and α-smooth muscle actin(ASMA), with the hepatocyte marker albumin (ALB) and possibly with oneor more other hepatocyte markers, preferably one or both of alpha-1antitrypsin and MRP2 transporter, and with at least one hepatic markerCD29 or alpha-fetoprotein (AFP).

Any said adult liver progenitor or stem cell may further express one,more than one, or all of the following molecules indicative ofhepatocyte-like properties or function: G6P, CYP1B1, CYP3A4, HNF-4, TDO,TAT, GS, GGT, CK8, EAAT2. The said adult liver progenitor or stem cellmay further be characterised by one, more than one, or all of thefollowing: negative for at least the hematopoietic markers CD45 and CD34and possibly also for one or more other hematopoietic markers, such as,e.g., CD105 & HLA-DR; negative for the cholangiocyte epithelial markercytokeratin-19 (CK-19) and possibly for more epithelial markers;negative for at least the undifferentiated stem cell markers CD117 andOct-4 and possibly also for one or more than one embryonic stem cellmarkers; low level expression of alpha-fetoprotein (AFP). Preferably,the said adult liver progenitor or stem cell may have mesenchymal-likemorphology, in particular involving one, more than one or all of growthin monolayers, flattened form, broad cytoplasm and/or ovoid nuclei withone or two nucleoli.

Identification of particular cell surface molecules by their CD (“commondeterminant”) designations is commonplace in the art. Other names ordesignations of molecules in the present application are also used aswell established in the art. Further specification of particularmolecules may be also found in the examples.

Wherein a cell is said to be positive for a particular marker, thismeans that a skilled person will conclude the presence or evidence of adistinct signal, e.g., antibody-detectable or detection possible byreverse transcription polymerase chain reaction, for that marker whencarrying out the appropriate measurement, compared to suitable controls.Where the method allows for quantitative assessment of the marker,positive cells may on average generate a signal that is significantlydifferent from the control, e.g., but without limitation, at least1.5-fold higher than such signal generated by control cells, e.g., atleast 2-fold, at least 4-fold, at least 10-fold, at least 20-fold, atleast 30-fold, at least 40-fold, at least 50-fold higher or even higher.

The expression of cell-specific markers can be detected using anysuitable immunological technique known in the art, such as flowcytometry, immuno-cytochemistry or affinity adsorption, Western blotanalysis, ELISA, etc., or by any suitable technique of measuring thequantity of the marker mRNA, e.g., Northern blot, semi-quantitative orquantitative RT-PCR, etc.

A skilled person will appreciate that the cell population obtained bythe present method, which comprises the above progenitor or stem cellsmay be harvested (e.g., by suitable detachment technique) and optionallyfurther enriched for cells displaying specific characteristics bymethods generally known in the art (hence, such cells can be isolatedfrom the said population). By means of illustration and not limitation,cells displaying one or more surface molecules characteristic of theprogenitor or stem cells of the invention, e.g., one or more of themarkers listed above, may be recognised by specific (labelled)antibodies or other recognition agents against such as markers andsorted out from cells not displaying such surface molecules, e.g., usingfluorescence activated cell sorting or using affinity binding to, e.g.,columns, beads or surfaces (panning). Any other ways of enrichment forthe cells are also included in the invention.

In a further aspect, the present inventors have thus realised a novel,isolated progenitor or stem cell (a vertebrate, preferably a mammal,even more preferably a human cell), originated from adult liver,characterised in that it co-expresses (i.e., is positive for) at leastone mesenchymal marker, esp. one, more than one, e.g., 2, 3, or 4, orall of the markers CD90, CD29, CD44, vimentin and α-smooth muscle actin(ASMA), with the hepatocyte marker albumin (ALB) and possibly with oneor more other hepatocyte markers. The said adult liver progenitor orstem cell may further be characterised by one, more than one, or all ofthe following: negative for at least the hematopoietic markers CD45,CD34 and CD117 and possibly also for one or more other hematopoieticmarkers; negative for cytokeratin-19 (CK-19); mesenchymal-likemorphology, in particular involving any or all of growth in monolayers,flattened form, broad cytoplasm and/or ovoid nuclei with one or twonucleoli.

Hence, in a particular embodiment (1), the isolated, liver-derivedprogenitor or stem cell co-expresses CD90, CD73, CD44, vimentin andα-smooth muscle actin (ASMA), with ALB. In a further embodiment (2), theliver-derived progenitor or stem cell co-expresses CD90, CD44, vimentinand α-smooth muscle actin (ASMA), with ALB and alpha-1 antitrypsin. In afurther embodiment (3), the liver-derived progenitor or stem cellco-expresses CD90, CD73, CD44, vimentin and α-smooth muscle actin(ASMA), with ALB and MRP2. In another embodiment (4), the liver-derivedprogenitor or stem cell co-expresses CD90, CD44, vimentin and α-smoothmuscle actin (ASMA), with ALB, alpha-1 antitrypsin and MRP2. In furtherembodiments (5), the liver-derived progenitor or stem cell of any of theabove embodiments (1) to (4) further express CD29. In furtherembodiments (6), the liver-derived progenitor or stem cell of any of theabove embodiments (1) to (4) further express alpha-fetoprotein. Infurther embodiments (7), the liver-derived progenitor or stem cell ofany of the above embodiments (1) to (4) further express CD29 andalpha-fetoprotein.

In further embodiments (8), the liver-derived progenitor or stem cell ofany of the above embodiments (1) to (7) are negative for CD45 and CD34.In further embodiments (9), the liver-derived progenitor or stem cell ofany of the above embodiments (1) to (7) are negative for CD117 andOct-4. In further embodiments (10), the liver-derived progenitor or stemcell of any of the above embodiments (1) to (7) are negative for CD45,CD34, CD117 and Oct-4.

In further embodiments (11), the liver-derived progenitor or stem cellof any of the above embodiments (1) to (10) are negative for CK19. Infurther embodiments (12), the liver-derived progenitor or stem cell ofany of the above embodiments (1) to (10) are negative for CK7. Infurther embodiments (13), the liver-derived progenitor or stem cell ofany of the above embodiments (1) to (10) are negative for CK19 and CK7.

In further embodiments (14), the liver-derived progenitor or stem cellof any of the above embodiments (1) to (13) show mesenchymal-likemorphology, preferably involving any or all of growth in monolayers,flattened form, broad cytoplasm and/or ovoid nuclei with one or twonucleoli.

In further embodiments (15), the liver-derived progenitor or stem cellof any of the above embodiments (1) to (14) further express one, morethan one, or all of the following molecules indicative ofhepatocyte-like properties or function: G6P, CYP1B1, CYP3A4, HNF-4, TDO,GS, GGT, CK8, EAAT2.

In further embodiments (16), the liver-derived progenitor or stem cellof any of the above embodiments (1) to (15) further express one, morethan one, or all of the following molecules: CD49e, CD13, CD54, majorhistocompatibility complex (MHC) class I (HLA-ABC).

In further embodiments (17), the liver-derived progenitor or stem cellof any of the above embodiments (1) to (16) are negative for one, morethan one, or all of the following molecules: CD105, HLA-DR, CD133,CD49b, CD49f & CD140.

In further embodiments (18), the liver-derived progenitor or stem cellof any of the above embodiments (6) to (17) express low level expressionof alpha-fetoprotein (AFP). Preferably, said low level correspondsessentially to the level of expression measured in normal hepatocytes.Preferably, it is less than the level of expression measured in thetumorigenic modified human liver cell line, e.g., HepG2.

The isolated, liver-derived progenitor or stem cell of the invention maypreferably display stem cell characteristics, esp. it may typicallydisplay at least limited (and possibly substantially unlimited)self-renewal, i.e., ability to propagate without differentiation. Bymeans of example and not limitation, the progenitor or stem cells of theinvention may be so propagated for at least 4 passages, e.g., at least 6passages, at least 10 passages, or at least 20 passages, at least 50passages, or more.

In another aspect, the invention provides an isolated adult liverprogenitor or stem cell, cell line thereof and/or a cell populationcomprising such, obtainable by or directly obtained using a methodcomprising: (a) disassociating, preferably by two-step collagenasemethod, adult liver or a part thereof from a subject, preferably avertebrate, mammal and more preferably a human subject, to form apopulation of primary cells from the said adult liver or part thereof;(b) plating the primary cell population onto collagen type I coatedsubstrate in Williams Medium E comprising foetal calf serum, preferably10% (v/v), EGF, preferably 25 ng/ml, insulin, preferably 10 μm/ml, anddexamethasone, preferably 1 μM; (c) allowing adherence of cells from theprimary cell population to the said substrate for 24 hours andthereafter exchanging the medium for fresh medium having composition asin (b); (d) culturing the cells in the said medium of (c) during twoweeks (preferably 15 days); (e) exchanging the medium for DMEMcomprising high glucose and FCS, preferably 10%, and further culturingthe cells, whereby the progenitor or stem cells of the invention emergeand proliferate; (f) optionally and preferably, allowing the cells tobecome about 70% confluent and passaging the cells at least once andpreferably at least two times, wherein the cells are plated onto thesubstrate as in (b) and cultured in a medium as in (e).

The step (a) may preferably include disassociation of the primary cellsby passing the cells through at least a sieve having pore size of 0.25mm, and preferably through a succession of sieves having graduallydecreasing pore sizes down to 0.25 mm, e.g., as in example 1.

The medium of step (e) may preferably not comprise dexamethasone,insulin and EGF and in an embodiment. The medium may in a furtherembodiment not comprise any exogenously added growth factor.

In an aspect, the invention also relates to the above methods.

In a further aspect, the invention provides an isolated liver progenitoror stem cell, cell line thereof and/or a cell population comprisingsuch, obtainable or directly obtained following the protocol set out inexample 1.

In a further aspect, the present inventors have established a cellpopulation (cell line) of adult human liver progenitor or stem cellsusing the methods of the invention, in particular as documented inexample 1, and deposited the said isolated cell line on Feb. 20, 2006under the Budapest Treaty with the Belgian Coordinated Collections ofMicroorganisms (BCCM) under accession number LMBP 6452CB (given by theInternational Depositary Authority; identification reference given bythe depositor: ADHLSC). Accordingly, in an aspect the present inventionrelates to an isolated cell, cell line and cell population depositedwith BCCM under accession number LMBP 6452CB (herein, the “LMBP 6452CB”cell line), sub-lines thereof including clonal sub-lines, and to progenythereof, including differentiated progeny thereof, esp. hepatocytes orhepatocytes-like cells prepared therefrom, and to genetically orotherwise modified derivatives thereof.

A skilled person will appreciate that progenitor or stem cells, celllines thereof and cell populations comprising such, obtainable accordingto the methods of the invention from adult human liver may havebiological properties, esp. proliferation and differentiation capacity,cell morphology and/or marker expression, identical or analogous to theabove deposited cell line, albeit they may be genetically different (dueto normal genetic variation between humans). Accordingly, a humanprogenitor or stem cell or cell population, esp. liver derived, havingbiological properties identical or analogous to the deposited cellpopulation is also encompassed in the present invention.

In a further aspect, the invention provides a cell population comprisingthe isolated adult liver progenitor or stem cells of the abovecharacteristics, and optionally further modified, e.g., geneticallymodified. In an embodiment, the said cell population may comprise about5% or more, e.g., about 10% or more of the said progenitor or stemcells, about 20% or more, about 30% or more, about 40% or more, about50% or more, about 60% or more, about 70% or more, about 80% or more orabout 90% or more of the progenitor or stem cells or is a substantiallyhomogeneous or homogeneous population of the said progenitor or stemcells.

In a further aspect, the invention provides a cell line established bypropagating the liver-derived progenitor or stem cells of the invention,optionally further modified, e.g., genetically modified. Suchpropagating may depart from one progenitor or stem cell (a clonal cellline) or from more than one cells.

In a yet further aspect, the invention provides an isolated adult liverprogenitor or stem cell, cell line thereof and/or a cell populationcomprising such, obtainable by or directly obtained using the abovedescribed methods of the invention, including preferred embodimentsthereof.

In a particular embodiment, the present invention thus provides a methodfor isolating a population of viable progenitor mesenchymal-like stemcells from adult normal (human) liver (FIG. 1). The stem cells were ableto proliferate in a culture medium, and expressed markers of severalliver cell types such as for instance albumin (hepatocytes), vimentin(stellate cells), alpha smooth muscle actin (ASMA) (FIG. 2A). They haveno biliary phenotype, as shown by negative immunostaining and RT-PCRassays for cytokeratin 19 (FIG. 2B). When tested using flow cytometry,ADHLSC were negative for CD45, CD34 and CD117 indicating theirnon-contamination by lymphohematopoietic lineage. In contrast, ADHLSCwere positive for CD90, CD29 and CD44, markers for mesenchymal lineage.

In an embodiment, the cells are able to retain their proliferationcapacity after trypsin/EDTA treatment. Incubation in a defined mediumallowed these cells to differentiate specifically into hepatocytes (FIG.3). As further specificity criteria, they were not able totrans-differentiate into osteocytes or adipocytes, as would be observedusing multipotent human mesenchymal bone marrow cells. Theirproliferation capacity, and their liver specificity lead to an increasedefficacy and safety for liver cell transplantation. Furthermore, becauseof their adult origin, these stem cells obviate the immunological,ethical and carcinogenic issues associated to embryonic cells.

Differentiation of the Progenitor or Stem Cells of the Invention

In addition to detecting cell markers, study of cells' differentiationin vitro and/or in vivo may be informative.

The present inventors further realised that the liver-derived progenitoror stem cells obtained using the above methods may possess specificdifferentiation capacity. In particular, the cells can be differentiatedinto hepatocytes or hepatocyte-like cells. In a yet further embodiment,the cells do not differentiate to mesodermal (mesenchymal) cell types,such as, e.g., osteocytes, chondrocytes, myocytes, connective tissuecells, tendonocytes, adipocytes or stromal cells.

The isolated liver-derived progenitor or stem cell of the invention,cell lines thereof or cell populations comprising such (specificallymentioning, albeit of course not limited to, the LMBP 6452CB line), orprogeny thereof, can be advantageously induced to differentiate to cellsof the hepatocyte lineage, in particular hepatocytes or hepatocyte-likecells. Such differentiation can occur in vivo, or in vitro or ex vivo.Accordingly, the invention further provides a method for generation ofhepatocytes or hepatocyte-like cells from the isolated progenitor orstem cell of the invention, and the resulting hepatocytes orhepatocyte-like cells.

In a particular embodiment, the liver-derived progenitor or stem cell ofthe invention is thus characterised by its ability to differentiate intohepatocytes or hepatocyte-like cells and lack of differentiation towardsmesodermal cell types, such as, e.g., osteocytes and adipocytes.

As understood by those skilled in the art, the ability to differentiatetowards a specific cell type or lack of such ability can be observed invitro, ex vivo or in vivo. By means of example, in vitro or ex vivodifferentiation can be assessed by exposing the cell to specificdifferentiation-inducing media as generally known in the art. Otherwise,differentiation can be assessed in vivo by following the fate of anintroduced (e.g., transplanted, injected, or otherwise administeredcell). A skilled person is able to recognise differentiation towardsparticular cell types by assessing phenotypic criteria including, butnot limited to, cell morphology, marker protein expression, and/oractivity of specific metabolic or other physiological pathways.

Differentiation into cells of the hepatocyte lineage may beadvantageously effected in the presence of cytokines and growth factors,which can be liver-specific. By means of illustration, hepatocyte growthfactor (HGF), or scatter factor, is a well-known cytokine that promotesdifferentiation to a hepatocyte phenotype. Similarly, a non-limitinglist of further cytokines includes epidermal growth factor (EGF), basicFGF, insulin, nicotinamide, oncostatin M, dexamethasone, HDAC inhibitors(e.g., sodium butyrate), DMSO, Vitamin A, or matrix components such asheparin sulphate, which have also been implicated in differentiationtowards hepatocytes. Protocols for inducing hepatocyte differentiationare generally known in the art and can be further optimised by a skilledperson.

Identification and subsequent isolation of differentiated cells fromtheir undifferentiated counterparts can be carried out by methods wellknown in the art. For example, cells that have been induced todifferentiate can be identified by selectively culturing cells underconditions whereby differentiated cells outnumber undifferentiatedcells. Similarly, differentiated cells can be identified bymorphological changes and characteristics that are not present on theirundifferentiated counterparts, such as cell size, shape or thecomplexity of intracellular organelle distribution. Also contemplatedare methods of identifying differentiated cells by their expression ofspecific marker proteins, such as cell-surface markers. Detection andisolation of these cells can be achieved, e.g., through flow cytometry,ELISA, and/or magnetic beads. Reverse-transcription polymerase chainreaction (RT-PCR) can also be used to monitor changes in gene expressionin response to differentiation. In addition, whole genome analysis usingmicroarray technology can be used to identify differentiated cells.

Genetic Modification of the Progenitor or Stem Cells of the Invention

The said adult liver progenitor or stem cell may be further modified,e.g., genetically modified as described above. The invention furtherrelates to the progeny, including differentiated progeny, of the adultliver progenitor or stem cell.

In an embodiment, to increase the replicative capacity of the obtainedprogenitor or stem cells of the invention, the cells can be telomerisedas generally known in the art. A cell is described as “telomerised” ifit has been genetically altered with a nucleic acid encoding atelomerase reverse transcriptase (TERT) of any species in such a mannerthat the TERT is transcribed and translated in the cell. The term alsoapplies to progeny of the originally altered cell that have inheritedthe ability to express the TERT encoding region at an elevated level.The TERT encoding sequence is typically taken or adapted from amammalian TERT gene, exemplified by human and mouse TERT, as indicatedbelow. Cells may be telomerised by genetically altering them with asuitable vector, so that they express the telomerase catalytic component(TERT) at an elevated level. Particularly suitable is the catalyticcomponent of human telomerase (hTERT), provided in WO 1998/14592. Forsome applications, other TERT sequences can be used. Other methods ofimmortalizing cells are also contemplated, such as genetically alteringthe cells with DNA encoding the SV40 large T antigen (U.S. Pat. No.5,869,243, WO 1997/32972), infecting with Epstein Bar Virus, introducingoncogenes such as myc and/or ras, introducing viral replication genessuch as adenovirus E1a, and fusing cells having the desired phenotypewith an immortalized cell line. Transfection with oncogenes or oncovirusproducts is usually less suitable when the cells are to be used fortherapeutic purposes.

In general, it might be preferred that the present progenitor or stemcells are not modified by telomerisation or other ways ofimmortalisation when the use of the said cells or progeny thereof,including differentiated progeny thereof, is contemplated in therapy,e.g., where such cells are to be introduced to human or animal, esp.human, body.

In the present invention, the obtained liver-derived progenitor or stemcells or progeny thereof may be stably or transiently transfected ortransformed with a nucleic acid of interest prior to further use, e.g.,in therapy or research, essentially as known in the art. Nucleic acidsequences of interest may include, but are not limited to, e.g., thoseencoding gene products which enhance the growth, differentiation and/orfunctioning of cell types useful in therapy, e.g., cell types derivablefrom the progenitor or stem cells of the invention, and particularly ofhepatocytes or hepatocyte like cells, or to deliver a therapeutic geneto a site of administration or implantation of such cells.

By means of example and not limitation, the obtained progenitor or stemcells or progeny thereof may be modified to constitutively or induciblyover-express a polypeptide normally expressed by liver cells, esp.hepatocytes, but being defective or absent in a patient, this defectunderlying a pathological state of the patient. Administration of the somodified cells may then restore production of the protein and therebyaid in treating the patient. For example, the progenitor or stem cellsor progeny thereof may contain heterologous DNA encoding a metabolicprotein such as ornithine transcarbamylase, argininosuccinatesynthetase, argininosuccinate lyase, arginase, carbamyl phosphatesynthase, N-acetyl glutamate synthase, glutamine synthetase, glycogensynthetase, glucose-6-phosphatase, succinate dehydrogenase, glucokinase,pyruvate kinase, acetyl CoA carboxylase, fatty acid synthetase, alanineaminotransferase, glutamate dehydrogenase, ferritin, low densitylipoprotein (LDL) receptor, P450 enzymes, and/or alcohol dehydrogenase.Alternatively, the cells may contain DNA encoding a secreted plasmaprotein such as albumin, transferrin, complement, component C3,α2-macroglobulin, fibrinogen, Factor XIII, Factor IX, α1-antitrypsin, orthe like.

The liver is a centre of production for many secretory proteins. It isanatomically connected with the circulatory system in such a way thatallows an efficient release of various proteins into the bloodstream.Therefore, genes encoding proteins that have systemic effects may beinserted into liver cells of the present invention as opposed to thespecific cell types that normally produce them, especially if it isdifficult to integrate genes into these cells. For example, a variety ofhormone genes or specific antibody genes may be inserted into livercells of the present invention for the secretion of their gene productsinto the circulation.

Conventional gene transfer methods are used to introduce nucleic acidsinto cells. The precise method used to introduce a gene is not criticalto the invention. For example, physical methods for the introduction ofDNA into cells include microinjection and electroporation. Chemicalmethods such as co-precipitation with calcium phosphate andincorporation of DNA into liposomes are also standard methods ofintroducing DNA into mammalian cells. Viral transfection is alsocontemplated. DNA is introduced using standard vectors, such as thosederived from murine and avian retroviruses (see, e.g., Gluzman et al.,Viral Vectors, Cold Spring Harbour Laboratory, Cold Spring Harbour,N.Y., 1988). Standard recombinant DNA methods are well known in the art(see, e.g., Ausubel et al., Current Protocols in Molecular Biology, JohnWiley & Sons, New York, 1989), and viral vectors for gene therapy havebeen developed and successfully used clinically (see, e.g., Rosenberg,et al., N. Engl. J. Med, 323:370 1990).

Uses of the Progenitor or Stem Cells of the Invention

The isolated, liver originated progenitor or stem cell of the invention,cell lines thereof or populations comprising such can be used fordifferent purposes (it is to be understood that the uses andapplications in the following are broadly contemplated for any adultliver progenitor or stem cell as defined above, in particular for cellsobtainable or directly obtained using the above defined methods, cellsdisplaying the above defined characteristics, as well as for the LMBP6452CB cell line, in a particular embodiment, an isolated adult derivedliver stem cell line, preferably an adult derived human liver stem cellline (ADHLSC); progeny thereof; or genetically modified derivativesthereof, including but not limited to:

-   -   liver cell transplantation in order to treat liver metabolic        deficiencies, liver degenerative diseases or fulminant liver        failure, using the isolated progenitor or stem cell or        population thereof according to the invention,    -   the preparation of bio-artificial liver devices using the        isolated progenitor or stem cell or population thereof according        to the invention,    -   the preparation of animal models of human liver diseases thanks        to transplantation of isolated progenitor or stem cell or        population thereof according to the invention in animals,    -   the preparation of in vitro and animal models of toxicology,        pharmacology using the isolated progenitor or stem cell or        population thereof according to the invention,    -   testing new drugs on the isolated progenitor or stem cell or        population thereof according to the invention, including        antiviral drugs for human hepatitis viruses.

For example, the analysis of the livers of uPA^(+/+)-SCID and SCID miceintrasplenically transplanted with ADHLSC (LMBP 6452CB line),demonstrated that these cells were able to engraft and to differentiateinto mature hepatocytes (FIGS. 4 & 5). Furthermore, human albumin wasdetected in the serum of these transplanted mice 10 weekspost-transplantation whereas no alpha-fetoprotein levels were detected.

According to the present invention the liver progenitor or stem cells(specifically mentioning, albeit of course not limited to, the LMBP6452CB line) can be used for the transplantation in inborn error ofmetabolism, for the transplantation in animal models of liver failure orfor the transplantation for animal models of human viral hepatitis. Thepresent cells can therefore be used in the treatment of liver associateddiseases including but not limited to liver failure, hepatitis, inbornerrors of metabolism.

In an exemplary embodiment, as shown in example 1, the inventorsdemonstrate that human liver progenitor or stem cells of the invention,when administered via intrasplenic injection to an immuno-deficientmammal, more preferably a rodent, esp. a mouse or rat, retain theirproliferation capacity and engraft within the host liver. Accordingly,in an embodiment, a cell population comprising liver progenitor or stemcells of the invention, preferably of human origin (specificallymentioning, albeit of course not limited to, the LMBP6452CB line), isintroduced, e.g., injected, and allowed to engraft in a mammalgenetically or chemically made immuno-deficient, to obtain an animalmodel. Preferably, such population may comprise at least 2×10⁶ cells ofthe invention. A skilled person will appreciate other ways ofadministering the cell population of the invention to induce liverengraftment of the said adult-derived liver progenitor or stem cells.

As detailed in example 1, the engrafted cells did not over-proliferate,thus underscoring their advantages in cell transplantation in humans orother animals, preferably mammals.

The present invention also encompasses the use of the isolatedprogenitor or stem cell or population thereof according to the invention(specifically mentioning, albeit of course not limited to, the LMBP6452CB line), for the following purposes:

-   -   liver progenitor or stem cell transplantation in order to treat        liver based inborn, metabolic deficiencies: Non exhaustive        examples of such diseases include phenylketonuria and other        aminoacidopathies, haemophilia and other clotting factor        deficiencies, familial hypercholesterolemia and other lipid        metabolism disorders, urea cycle disorders, glycogenosis,        galactosemia, fructosemia, tyrosinemia, protein and carbohydrate        metabolism deficiencies, organic aciduria, mitochondrial        diseases, peroxysomal and lysosomal disorders, protein synthesis        abnormalities, defects of liver cell transporters, defect of        glycosylation and the like,    -   transplantation of the liver progenitor or stem cell according        to the invention to treat acquired progressive liver        degenerative diseases,    -   use of the liver progenitor or stem cell according to the        invention to treat fulminant liver failure and acute or chronic        liver failure,    -   use of the liver progenitor or stem cell according to the        invention in bio-artificial liver devices and liver assist        devices,    -   animal models of human liver diseases thanks to transplantation        of the liver progenitor or stem cell according to the invention        in small and large animals,    -   the preparation of animal models of human hepatotropic virus        infections (HBV, HAV, HCV, HEV, HDV, . . . ) to study natural        history, transmission, resistance, effects of treatment, use of        antiviral drugs or any research using transplanted the liver        progenitor or stem cell according to the invention,    -   the preparation of in vitro and animal models of toxicology,        pharmacology and pharmacogenetics using the liver progenitor or        stem cell according to the invention,    -   the testing new drugs on the liver progenitor or stem cell        according to the invention,    -   gene therapy, by inserting viral sequences in the liver        progenitor or stem cell according to the invention which can        then be expanded in vitro,    -   animal models to study human liver cell metabolism,    -   tolerance of allogeneic cells thanks to the use of the liver        progenitor or stem cell according to the invention, and/or    -   use of the liver progenitor or stem cell according to the        invention to avoid, prevent or treat liver or liver cell        allograft rejection.

The present progenitor or stem cells or progeny thereof, includingdifferentiated progeny, may in an aspect of the invention be intendedfor therapeutic applications, e.g., for tissue engineering and celltherapy.

A skilled person will appreciate that the herein detailed uses mayinvolve the use of the progenitor or stem cells, cell lines thereof,cell populations comprising such, as well as the use of progeny thereof,including differentiated progeny, esp. hepatocytes or hepatocyte-likecells, or genetically modified derivatives thereof.

As noted above, the liver originated progenitor or stem cells of theinvention, cell lines thereof or cell populations comprising such(specifically mentioning, albeit of course not limited to, the LMBP6452CB line), or progeny thereof, optionally genetically modified, canbe used in cell replacement therapies. The cells can be administered toa tissue of interest in a subject to supplement functioning cells orreplace cells, which have lost function. Alternatively, methods ofproviding differentiated cells, particularly hepatocytes orhepatocyte-like cells, are also contemplated, wherein progenitor or stemcells are differentiated in the presence of differentiation factors,isolated, and administered in a subject.

Disease states or deficiencies typified by loss of liver mass and/orfunction, and that could benefit from progenitor or stem cells of theinvention include those listed above, and further include but are notlimited to Alagille syndrome, alcoholic liver disease (alcohol-inducedcirrhosis), a1-antitrypsin deficiency (all phenotypes), hyperlipidemiasand other lipid metabolism disorders, autoimmune hepatitis, Budd-Chiarisyndrome, biliary atresia, progressive familial cholestasis type I, IIand III, cancer of the liver, Caroli Disease, Crigler-Najjar syndrome,fructosemia, galactosemia, carbohydrate deficient glycosylation defects,other carbohydrate metabolism disorders, Refsum disease and otherperoxysomal diseases, Niemann Pick disease, Wolman disease and otherlysosomal disorders, tyrosinemia, triple H, and other amino acidmetabolic disorders, Dubin-Johnson syndrome, fatty liver nonalcoholicsteatohepatitis), Gilbert Syndrome, Glycogen Storage Disease I and III,hemochromatosis, hepatitis A-G, porphyria, primary biliary cirrhosis,sclerosing cholangitis, tyrosinemia, clotting factor deficiencies,hemophilia B, phenylketonuria, Wilson's Disease, fulminant liverfailure, post hepatectomy liver failure, mitochondrial respiratory chaindiseases. In addition, the cells can also be used to treat acquiredliver disorders due to viral infections.

Accordingly, in an aspect are provided adult liver progenitor or stemcells of the invention, cell lines thereof or cell populationscomprising such (specifically mentioning, albeit not limited to, theLMBP 6452CB line), or progeny thereof including differentiated progeny,esp. hepatocytes or hepatocyte like cells, optionally geneticallymodified as detailed above, for use in therapy and/or use thereof forthe manufacture of a medicament for the treatment of liver diseases.Such diseases may include disorders affecting liver tissue, diseasesaffecting the hepatocyte viability and/or function are specificallycontemplated, and may represent, e.g., inborn errors, the effect of adisease condition, the effect of trauma, toxic effects, viralinfections, etc. Liver diseases listed in the present specification arespecifically contemplated. Administration of the cells according to theinvention can lead to tissue reconstitution or regeneration in thesubject. The cells are administered in a manner that permits them tograft or migrate to the intended tissue site and reconstitute orregenerate the functionally deficient area.

Another aspect of the invention is a method for preventing and/ortreating a liver disease, comprising administration of adult liverprogenitor or stem cells of the invention, cell lines thereof or cellpopulations comprising such (specifically mentioning, albeit not limitedto, the LMBP 6452CB line), or progeny thereof including differentiatedprogeny, esp. hepatocytes or hepatocyte like cells, optionallygenetically modified, to a subject, esp. human, in need of suchtreatment. Such administration is typically in therapeutically effectiveamount, i.e., generally an amount which provides a desired local orsystemic effect and performance.

In a further aspect, the invention relates to a pharmaceuticalcomposition comprising the adult liver progenitor or stem cells of theinvention, cell lines thereof or a cell population comprising such(specifically mentioning, albeit not limited to, the LMBP 6452CB line),or progeny thereof including differentiated progeny, esp. hepatocytes orhepatocyte like cells, optionally genetically modified as above.

By means of example and not limitation, the isolated liver progenitor orstem cells of the invention, cell lines thereof or cell populationscomprising such (specifically mentioning, albeit of course not limitedto, the LMBP 6452CB line), or progeny thereof can be advantageouslyadministered via injection (encompassing also catheter administration)or implantation, e.g. localised injection, systemic injection,intrasplenic injection (see also Gupta et al., Seminars in Liver Disease12: 321, 1992), injection to a portal vein, injection to liver pulp,e.g., beneath the liver capsule, parenteral administration, orintrauterine injection into an embryo or foetus.

In a preferred embodiment, the liver originated progenitor or stem cellsof the invention, cell lines thereof or cell populations comprising such(specifically mentioning, albeit of course not limited to, the LMBP6452CB line), or progeny thereof, optionally genetically modified, canbe used for tissue engineering and cell therapy via liver celltransplantation (LCT). Liver cell transplantation, and liver stem celltransplantation (LSCT) refers to the technique of infusing maturehepatocytes or liver progenitor cells, including the cells of theinvention, in any way leading to hepatic access and engraftment of thecells, preferably via the portal vein, but also by direct hepaticinjection, or by intrasplenic injection.

For example, the cells may be provided as a cell suspension in anypreservation medium, preferably containing human albumin, afterisolation procedure or after thawing following cryopreservation.

In an embodiment, the present invention contemplates using a patient'sown liver tissue to isolate the progenitor or stem cells of theinvention. Such cells would be autologous to the patient and could bereadily administered to the patient. Moreover, if the patient containeda genetic defect underlying a particular pathological condition, suchdefect could be averted by genetically manipulating the obtained cells.

In another embodiment, the progenitor or stem cells of the invention maybe isolated from tissue which is not patient's own. Where administrationof such cells to a patient is contemplated, it may be preferable thatthe liver tissue subjected to the method of the present invention toobtain the progenitor or stem cells, is selected such as to maximise, atleast within achievable limits, the tissue compatibility between thepatient and the administered cells, thereby reducing the chance ofrejection of the administered cells by patient's immune system (e.g.,graft vs. host rejection).

The ability of the immune system to differentiate self from non-self isto a large extent determined by products of the major histocompatibilitycomplex (MHC), whose genes are on chromosome 6 and belong to theimmunoglobulin gene super-family. Class I WIC products consist of HLA-A,HLA-B and HLA-C; these have a wide distribution and are present on thesurface of essentially all nucleated cells and on platelets. Class IIWIC products consist of HLA-D, HLA-DR, HLA-DP, and HLA-DQ; they have amore limited distribution, including on B cells, macrophages, dendriticcells, Langerhans' cells, and activated (but not resting) T cells.

The HLA loci are generally multi-allelic, e.g., using specificantibodies, at least 26 HLA-A alleles, 59 HLA-B alleles, 10 HLA-Calleles, 26 HLA-D alleles, 22 HLA-DR alleles, nine HLA-DQ alleles andsix HLA-DP alleles can be recognized. Because HLA loci are closelylinked, the HLA antigens may also be present as conserved haplotypes.

A subject in need of therapy with cells of the present invention may bescreened for the presence of anti-HLA antibodies and for his HLAgenotype and/or phenotype (e.g., on lymphocytes; e.g., using serologicalmethods or genetic DNA analysis). Progenitor or stem cells obtainableaccording to the present invention, or the source liver tissue or thedonor thereof, may be typically tested for their HLA phenotype and/orgenotype and suitable tissues or cells selected for administration,which have either identical HLA haplotypes to the patient, or which havethe most HLA antigen alleles common to the patient and none or the leastof HLA antigens to which the patient contains pre-existing anti-HLAantibodies. The probability that the transplanted cells will besuccessfully accepted increases with the number of identical HLAantigens. A skilled person will understand the further variations ofthese considerations.

Other ways of obtaining MHC profile resembling the patient's are alsocontemplated, e.g., genetic manipulation of the obtained progenitor orstem cells of the invention or progeny thereof.

If the cells are derived from heterologous (i.e., non-autologous)source, concomitant immunosuppression therapy may be typicallyadministered, e.g., using immunosuppressive agents, such as cyclosporineor tacrolimus (FK506). Alternatively, the cells can be encapsulated in amembrane which permits exchange of fluids but prevents cell/cellcontact. Transplantation of microencapsulated cells is known in the art,e.g., Balladur et al., 1995, Surgery 117:189-194; and Dixit et al.,1992, Cell Transplantation 1:275-279. Preferably, the cells may beautologous, or displaying a close HLA match as explained.

In another preferred embodiment, the adult liver or part thereof is froma non-human animal subject, preferably a non-human mammal subject.Progenitor or stem cells or cell lines, or progeny thereof, derivedaccording to the invention from livers of non-human animal or non-humanmammal subjects can be advantageously used, e.g., in research and in thetherapy of liver disease in members of the same, related or othernon-human animal or non-human mammal species, or even in the therapy ofhuman patients suffering from liver disease (e.g., xenotransplantation,bio-artificial liver devices comprising non-human animal or non-humanmammal cells). By means of example and not limitation, particularlysuitable non-human mammal cells for use in human therapy may originatefrom pigs.

An issue concerning the therapeutic use of the progenitor or stem cellsof the invention is the quantity of cells necessary to achieve anoptimal effect. Doses for administration may be variable, may include aninitial administration followed by subsequent administrations; and canbe ascertained by the skilled artisan armed with the present disclosure.Typically, the administered dose or doses will provide for atherapeutically effective amount of the cells, i.e., one achieving thedesired local or systemic effect and performance.

In current human studies of autologous mononuclear bone marrow cells,empirical doses ranging from 1 to 4×10⁷ cells have been used withencouraging results. However, different scenarios may requireoptimisation of the amount of administered cells. Thus, the quantity ofcells to be administered will vary for the subject being treated. In apreferred embodiment, between 10² to 10⁹, or between 10³ to 10⁹, orbetween 10⁴ to 10⁹, such as between 10⁴ and 10⁸, or between 10⁵ and 10⁷,e.g., about 1×10⁵, about 5×10⁵, about 1×10⁶, about 5×10⁶, about 1×10⁷,or about 2×10⁷, about 3×10⁷, about 4×10⁷, about 5×10⁷, about 6×10⁷,about 7×10⁷, about 8×10⁷, about 9×10⁷, or about 1×10⁸, cells can beadministered to a human subject. However, the precise determination of atherapeutically effective dose may be based on factors individual toeach patient, including their size, age, size tissue damage, and amountof time since the damage occurred, and can be readily ascertained bythose skilled in the art from this disclosure and the knowledge in theart.

Preferably, purity of a cell population comprising the progenitor orstem cells of the invention for administration may be about 50 to about55%, about 55 to about 60%, and about 65 to about 70%. More preferablythe purity may be about 70 to about 75%, about 75 to about 80%, about80% to about 85%; and most preferably the purity may be about 85 toabout 90%, about 90 to about 95%, and about 95 to about 100%. Purity ofthe stem cells can be determined, e.g., according to the cell surfacemarker profile within a cell population. Dosages can be readily adjustedby those skilled in the art (e.g., lower purity may require an increasein dosage).

The skilled artisan can readily determine the amount of cells andoptional additives, vehicles, and/or carrier in compositions to beadministered in methods of the invention. Typically, any additives (inaddition to the active progenitor or stem cell(s) and/or cytokine(s))may be present in an amount of 0.001 to 50% (w/w or w/v) solution inphosphate buffered saline, and the active ingredient may be typicallypresent in the order of micrograms to milligrams, such as about 0.0001to about 5% (w/w or w/v), preferably about 0.0001 to about 1%, mostpreferably about 0.0001 to about 0.05% or about 0.001 to about 20%,preferably about 0.01 to about 10%, and most preferably about 0.05 toabout 5%.

When administering a therapeutic composition of the present invention,it may generally be formulated in a unit dosage injectable form (e.g.,solution, suspension, dispersion, emulsion). The pharmaceuticalformulations suitable for injection include sterile aqueous solutionsand dispersions. As used herein, the solutions or dispersions include apharmaceutically acceptable carrier or diluent in which the cells of theinvention remain viable. The carrier can be a pharmaceuticallyacceptable solvent or dispersing medium containing, for example, water,saline, phosphate buffered saline, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol, and the like) and suitablemixtures thereof.

Additionally, various additives which enhance the stability, sterility,and isotonicity of the compositions, including antimicrobialpreservatives, antioxidants, chelating agents, and buffers, can beadded. Prevention of the action of microorganisms can be ensured byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, and the like.

In many cases, it will be desirable to include isotonic agents to ensureviability of the cells, for example, sugars, sodium chloride, and thelike. The desired isotonicity of the compositions of this invention maybe accomplished using sodium chloride, or other pharmaceuticallyacceptable agents such as dextrose, boric acid, sodium tartrate,propylene glycol or other inorganic or organic solutes. Sodium chlorideis preferred particularly for buffers containing sodium ions.

To potentially increase cell survival when introducing the progenitor orstem cells or differentiated progeny of interest thereof into a subjectin need thereof, may be to incorporate the said cells into a biopolymeror synthetic polymer. Examples of suitable biopolymers include, but arenot limited to, fibronectin, fibrin, fibrinogen, thrombin, collagen, andproteoglycans. This could be constructed with or without includedcytokines, growth factors, differentiation factors or nucleic acidexpression constructs, etc. Such biopolymers could be, e.g., insuspension or could for a three-dimensional gel with the cells embeddedthere within. Such polymers can be preferably biodegradable.

Prolonged absorption of the injectable pharmaceutical form can bebrought about by the use of agents delaying absorption, for example,aluminum monostearate and gelatin. According to the present invention,however, any vehicle, diluent, or additive used would have to becompatible with the progenitor or stem cells.

Sterile injectable solutions can be prepared by incorporating the cellsutilized in practicing the present invention in the required amount ofthe appropriate solvent with various amounts of the other ingredients,as desired.

Such compositions may further be in admixture with a suitable carrier,diluent, or excipient such as sterile water, physiological saline,glucose, dextrose, or the like. The compositions can contain auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,gelling or viscosity enhancing additives, preservatives, flavouringagents, colours, and the like, depending upon the route ofadministration and the preparation desired.

Standard texts, such as “Remington's pharmaceutical science”, 17thedition, 1985, incorporated herein by reference, may be consulted toprepare suitable preparations, without undue experimentation.

Viscosity of the compositions, if desired, can be maintained at theselected level using a pharmaceutically acceptable thickening agent.Methylcellulose is preferred because it is readily and economicallyavailable and is easy to work with. Other suitable thickening agentsinclude, for example, xanthan gum, carboxymethyl cellulose,hydroxypropyl cellulose, carbomer, and the like. The preferredconcentration of the thickener will depend upon the agent selected. Thepoint is to use an amount, which will achieve the selected viscosity.Viscous compositions are normally prepared from solutions by theaddition of such thickening agents.

Further Uses of the Progenitor or Stem Cells of the Invention

Further uses of the progenitor or stem cells of the invention, celllines thereof or cell populations comprising such (specificallymentioning, albeit not limited to, the LMBP 6452CB line), or progenythereof including differentiated progeny, esp. hepatocytes or hepatocytelike cells, optionally genetically modified, are contemplated below.

Thus, the progenitor or stem cells or differentiated derivativesthereof, esp. hepatocytes or hepatocyte like cells, can be used todetect cellular responses (e.g., toxicity) to bioactive (biologic orpharmacologic) agents, comprising contacting a culture of cells, or thedifferentiated derivatives thereof, with one or more biologic orpharmacologic agents, identifying one or more cellular response to theone or more biologic or pharmacologic agents, and comparing the cellularresponses of the cell cultures to the cellular responses of controlcultures. Such responses can be determined by monitoring the activitiesof molecules such as, but not limited to, alkaline phosphatase,cytochrome P450, urea pathway enzymes, among others.

Further, cytokines, chemokines, pharmaceutical compositions and growthfactors, for example, can be screened using the progenitor or stem cellof the invention or differentiated derivatives thereof, esp. hepatocytesor hepatocyte like cells, to more clearly elucidate their effects ondifferentiation and function of such cells.

The invention also envisions a tissue-engineered organ, or portion, orspecific section thereof, a tissue engineered device comprising a tissueof interest and optionally, cytokines, growth factors, ordifferentiation factors, wherein the cells of the invention are used togenerate tissues, esp. liver tissue, esp. tissues comprisinghepatocytes. Tissue-engineered organs can be used with a biocompatiblescaffold to support cell growth in a three-dimensional configuration,which can be biodegradable. Tissue-engineered organs generated from thestem cells of the present invention can be implanted into a subject inneed of a replacement organ, portion, or specific section thereof. Thepresent invention also envisions the use of the stem cells or cellsdifferentiated therefrom as part of a bioreactor, e.g., a liver assistdevice.

Organs, portions, or sections derived from the stem cells of theinvention can be implanted into a host. The transplantation can beautologous, such that the donor of the stem cells from which organ ororgan units are derived is the recipient of the engineered tissue. Thetransplantation can be heterologous, such that the donor of the stemcells from which organ or organ units are derived is not that of therecipient of the engineered-tissue. Once transferred into a host, thetissue-engineered organs can recapitulate the function and architectureof the native host tissue. The tissue-engineered organs will benefitsubjects in a wide variety of applications, including the treatment ofcancer and other disease disclosed herein, congenital defects, or damagedue to surgical resection.

As a tool for the drug testing and development process, the liver cellsand their progeny could be used to assess changes in gene expressionpatterns caused by drugs being considered for development. The changesin gene expression pattern from potential drugs could be compared withthose caused by drugs known to affect the liver. This would allow apharmaceutical company to screen compounds for their effect on the liverearlier in the development process, saving time and money. The fulllineage of liver cells, from progenitors to mature cells, could also beused to test drugs for toxicity to the liver and to study how the drugis metabolized. Currently, pharmaceutical companies have difficultyobtaining a consistent supply of liver cells for toxicity testing. Themethods and cells of the present invention answer this need.

Further, the adult liver progenitor or stem cells of the invention orprogeny thereof including differentiated progeny, esp. hepatocytes orhepatocyte like cells are useful as biological components ofdetoxification devices such as liver perfusion or liver assist devices.

A conventional liver assist device includes a rigid, plastic outer shelland hollow semi-permeable membrane fibres which are seeded with stemcells or differentiated hepatocytes or derived from the stem cells. Thefibres may be treated with collagen, lectin, laminin, or fibronectin,for the attachment of cells or left untreated. Bodily fluid is perfusedthrough the device for detoxification according to well known proceduresand then returned to the patient. An example of an LAD suitable for thecells of the present invention is described in International PatentPublication Serial Number PCT US00/15524.

The adult liver progenitor or stem cells of the invention or progenythereof can be differentiated in vitro and further used in place ofmature hepatocytes in “ADMET” administration, distribution, metabolism,elimination and toxicology) or cytotoxicity tests.

Hepatocytes or hepatocyte-like cells obtained by differentiating theadult liver progenitor or stem cells of the invention or progeny thereofmay provide an in vitro model for liver development study, liver cellmetabolism or liver cell biology; and for screen differentiating,proliferative or toxic molecules. Further contemplating geneticmanipulation of such cells, they can be used to study genes implicatedin liver development, liver cell metabolism or biology.

The invention will now be illustrated by means of the followingexamples, which do not limit the scope of the invention in any way.

Example 1

Liver Cell Isolation Procedure

Human liver cells were obtained from the whole liver or liver segmentsoriginating from healthy cadaveric or non heart beating donors. Cellswere isolated after the gross clamp time (e.g., between 6 and 12 hoursafter gross clamp time) while livers were kept on ice in a University ofWisconsin medium until the perfusion. Hepatocytes were isolated using adassic 2-step perfusion technique {Seglen, 1976} {Stephenne, 2005}.Liver tissue was sequentially perfused by the apparent blood vesselswith an EGTA solution (Earl's Balanced Salt Solution without Ca⁺⁺ andMg⁺⁺, 0.5 mM EGTA, 5 mM Hepes, 2 mg/l gentamicin, and 100,000 IU/lpenicillin G) and a digestion enzyme solution for 9 to 12 minutes eachat 37° C. The digestion solution (EBSS with Ca⁺⁺ and Mg⁺+, 5 mM Hepes, 2mg/l gentamicin, and 100,000 IU/l penicillin G) included 0.9 mg/ml ofcollagenase P and 0.03 mg/ml of soybean trypsin inhibitor. The livercapsule was incised and the hepatocytes were released by gentle shaking.Digestion Was stopped with ice-cold wash medium (medium M199, 5 mMHepes, 2 mg/l gentamicin and 100,000 IU/l penicillin G), containing 0.03mg/ml of soybean trypsin inhibitor and 100 ml/l of human plasma. Thecells were filtered and rinsed through 4 metal sieves of respectively,4.5 mm, 1 mm, 0.5 mm, and 0.25 mm of pore size. Cells were washed 3times by centrifugation at 1200 rpm for 3 minutes in a cold M199 washmedium.

Primary Cell Culture

Single cell suspensions were resuspended in Williams' E medium(Invitrogen) supplemented with 10% fetal calf serum (FCS) (Perbio,Hyclone), 25 ng/ml EGF (Peprotech), 10 μm/ml insulin, 1 μM dexamethazoneand 1% penicillin/streptomycin (P/S) (Invitrogen). The cells were platedon rat tail collagen I (BD Biosciences)—coated flasks or plates (e.g.,6-well plates) (Greiner Bio-one) and cultured at 37° C. in a fullyhumidified atmosphere containing 5% CO₂. After 24 hours, medium waschanged in order to eliminate the non-adherent cells and thereafterrenewed every three days. During two weeks, the culture was followedmicroscopically everyday and culture medium was analyzed every threedays. Culture medium was then switched to DMEM with high glucoseconcentrations (Invitrogen) supplemented with 10% FCS (Perbio, Hyclone)and 1% P/S (Invitrogen) in order to accelerate the elimination of adulthepatocytes. A cell type with mesenchymal-like morphology thenspontaneously emerged, proliferated and filled the empty space in thewell plate as confirmed by phase contrast microscopy. These cellsappeared between day 15 and 20 of the culture and presented a flattenedform, broad cytoplasm and ovoid nuclei with one or two nucleoli (FIG.1). When reaching 70% confluence, cells were lifted with 0.25% trypsinand 1 mM EDTA and re-plated at the desired concentration. The analysisof cell suspension using flow cytometry showed that the populationbecame homogenous after passage 2. For each passage, cell suspension wasalso analyzed using RT-PCR, and immunofluorescence.

Characterization of the Cells

In order to avoid the possible contamination by other cell types,immunocytochemistry was performed to study the phenotype of these cells.Because of their hepatic origin, expression of specific markers such asalbumin has been analyzed in paraformaldehyde-fixed cells. As shown inFIG. 2, albumin which is exclusively expressed in hepatocytes, wasdetected both using monoclonal (Sigma clone HAS-111) or polyclonal(Chemicon) antibodies. In parallel, expression of mesenchymal cellmarkers has also been evaluated demonstrating that these cells areimmuno-positive for vimentin and alpha smooth muscle actin (FIG. 2). Sofar the phenotypic characteristics have been studied over 7 passageswith great stability.

Cell Differentiation:

Cells were seeded at a density of 0.5-1×10⁴/cm² in 6 well plates coatedwith rat-tail collagen type I in DMEM supplemented with FCS and P/S.Culture medium was switched 24 hours later to Iscove's modifiedDulbecco's medium (IMDM) (Invitrogen). For induction, the cells wereincubated for 2 weeks with induction medium containing IMDM supplementedwith 20 ng/ml HGF (Biosource), 10 ng/ml bFGF (Peprotech) and 0.61 g/lnicotinamide (Sigma). Thereafter, cells were incubated with maturationmedium containing IMDM supplemented with 20 ng/ml oncostatin M (Sigma),1 μM dexamethasone (Sigma), 50 mg/ml ITS (insulin, transferrin,selenium) (Invitrogen). For induction and maturation steps, medium waschanged and analyzed every 3 days. After exposure to these cocktails,cells started to lose their sharp edges, were progressively shrunk andlost their initial morphology to adopt a polygonal shape (FIG. 3).

Flow Cytometry:

Cells were collected after centrifugation at 1200 rpm for 5 min andre-suspended at a concentration of 500 to 1000/μl in PBS. Cells werethen incubated for 30 minutes at 4° C. with antibodies. Thecorresponding control isotypes were used for evaluation of nonspecificbinding of monoclonal antibodies. Cells were then washed and resuspendedin Isoton® (Beckham Coulter) for reading with a Beckham Coulter FlowCytometer.

RT-PCR

Total RNA was extracted from cells grown in 6 wells-plates using theTriPure isolation reagent (Roche) and cDNA was generated using thereverse transcription kit, according to the manufacturer's instructions.PCR amplifications were performed using polymerase elongase in a finalvolume of 25 μl and appropriate primers. Samples were thereafterelectrophoresed on a 1% agarose gel and nucleic acids were visualized byethidium bromide staining.

Immunofluorescence

For immunostaining, cells grown on rat tail collagen I-coated 12 mmround glass coverslips were fixed with paraformaldehyde 4% (v/v) for 15min at room temperature and permeabilised thereafter with 1% Triton X100(v/v) in TBS (Tris-HCl 50 mM, NaCl 150 mM, pH 7.4) during 15 min.Non-specific immunostaining was prevented by 1 h incubation in a TBSsolution containing 3% non-fat dry milk at 37° C. Cells were thensuccessively incubated in the same solution for 1 h at room temperaturewith primary antibodies, rinsed 5 times with TBS and for 1 h withsecondary antibodies (1/500). Nuclei were stained during 30 min with thenuclear dye DAPI (1/5,000). After 3 rinses, preparations were mounted inFluoprep medium (BioMerieux, Brussels, Belgium) and examined using anOlympus IX70 inverted microscope coupled to a CCD camera (T.I.L.L.photonics, Martinsried, Germany). Excitation light (552, 488 and 372 nmfor Cy-3, FITC and DAPI, respectively) was obtained from a Xenon lampcoupled to a monochromator (T.I.L.L. photonics, Martinsried, Germany).Digital images were acquired using appropriate filters and combinedusing the TILLvision software.

Molecules Detected in the Progenitor or Stem Cells of the Invention

Using the above approaches, the following expression profile of a numberof cell markers has been established in an experiment for the cellsestablished in this example (ADHLSC):

CD90 positive. CD90, or Thy-1, is a cell-surface protein, considered tobe indicative of mesenchymal lineage.

CD44 positive. CD44 is a cell-adhesion molecule and is used to identifyat least some types of mesenchymal stem cells (MSC).

Vimentin positive. Vimentin is a type III intermediate filament commonlydetected in mesenchymal cells and fibroblasts.

Albumin positive. Albumin is a plasma protein produced and secreted bythe liver. Within hepatocytes, albumin is usually found as a cytoplasmicprotein.

CD29 positive. CD29, also known as integrin beta-1, is a transmembraneglycoprotein, also present in hepatic tissue, thought to form withintegrin alpha a functional receptor complex involved in interactionwith extracellular matrix.

CD73 positive. CD73 is an ecto 5′-nucleotidase considered to be amesenchymal marker.

CD49b positive. CD49b is also called integrin alpha-2 or collagenreceptor and is implicated in cell interaction with extracellularmatrix.

HLA-ABC positive. HLA-ABC (human leukocyte antigens A, B & C) are majorhistocompatibility complex class I antigens forming membraneheterodimers.

Alpha-fetoprotein—low levels of expression. Alpha-fetoprotein is aprotein expressed during development of primitive endoderm andthroughout maturation, reflects endodermal lineage. High levels ofalpha-fetoprotein expression usually reveals tumorigenic shunt.

Alpha-1 antitrypsin positive. Alpha-1 anti trypsin is a plasma proteinsynthesized by the liver.

Glucose 6-phosphatase (G6P) positive. G6P is a liver enzyme thathydrolyzes glucose 6-phosphate to glucose and inorganic phosphate,allowing glucose in the liver to enter the blood.

Cytochrome P450 1B1 (CYP1B1) positive. CYP1B1 is a dioxin induciblecytochrome responsible for the phase I metabolism of a wide range ofstructurally diverse substrates.

Cytochrome P450 3A4 (CYP3A4) positive. CYP3A4 is a crucial enzymeinvolved in the metabolism of xenobiotics.

Hepatocyte Nuclear Factor 4 (HNF-4) positive. HNF4, a nuclear receptor,is a transcription factor involved in the regulation of energymetabolism.

Tryptophan 2,3-dioxygenase (TDO) positive. TDO is the first enzymeinvolved in tryptophan oxidation in the liver.

Tyrosine aminotransferase (TAT) positive. TAT is a mitochondrial liverspecific enzyme involved in amino acid metabolism and gluconeogenesis

Glutamine synthase (GS) positive. GS is a key enzyme for ammoniumassimilation.

Gamma glutamyl transpeptidase (GGT) positive. GGT is an enzyme involvedin the metabolism of glutathione.

Cytokeratin 8 (CK8) positive. CK8 is an intermediate filament specificof epithelial cells.

Multi-drug resistant protein 2 (MRP2) positive. MRP2 is an organ aniontransporter responsible for the export of intracellular organic anionsfrom hepatocytes to the biliary tree.

Glutamate transporter 2 (EAAT2) positive.

The presence of the above many molecules that may participate in liverfunction and metabolism is indicative of the close link of the ADHLSCcell line to liver phenotypes.

CD117 negative. CD117, also called c-kit, is a cell-surface receptor onbone marrow cell types that identifies HSC and MSC thereforecharacterize quite undifferentiated stem cells.

CD34 negative. CD34 is a cell surface protein on bone marrow cells,indicative of HSC and endothelial progenitor.

CD45 negative. CD45, also called leukocyte antigen, is a tyrosinephosphatase expressed cells of the hematopoietic lineage, includinghematopoietic stem cells.

CD105 negative. CD105, also called SH2 or endoglin, is an adhesionmolecule. It is also considered to be a marker of mesenchymal stemcells.

CD133 negative. CD133 is a hematopoietic stem cell marker.

HLA-DR negative. These major histocompatibility complex class IIantigens are membrane heterodimers restrictively expressed in antigenpresenting cells.

Oct-4 negative. Oct-4 is a transcription factor expressed only bypluripotent stem cells and essential for maintenance of theundifferentiated state.

Cytokeratin 19 (CK19) negative. CK19 is widely used as a marker forbiliary cells, i.e., cholangiocytes.

Cytochrome P 2B6 (CYP2B6) negative. CYP2B6 is involved in the metabolismof endo- and xenobiotics.

CD54: Intercellular Adhesion Molecule-1 (ICAM-1), a membraneglycoprotein.

Without intending to be limiting in any way, the present inventors,based on their knowledge of cell markers, put forward a followingpossible interpretation of the above data: This combination of markersdefines an original cell line, which expresses markers from themesenchymal lineage (CD90, CD73, vimentin, CD44) as well as markerscharacteristic of the hepatic differentiation path (CD29 and albumin,alpha-1 antitrypsin, HNF4, MRP2 transporter). The presence of albumindetected by both immuno-fluorescence and RT-PCR strongly argue against apossible contamination with stellate cells. The ADHLSC seem not to bepluripotent undifferentiated mesenchymal stem cells (CD45 negative, CD34negative, CD117 negative, Oct-4 negative), nor liver stellate cells(albumin positive). The ADHLSC line seems committed to the hepaticlineage (CD29 positive, expressing albumin and alpha-1 anti trypsin) butdoes not express typical biliary marker (CK19 and 7 negative). Hence,the cells and cell lines of the invention may, in one and not limitingway, be denoted as mesenchymal stem cell line with characteristics of ahepatocyte progenitor.

uPA-SCID Mice Transplantation, Histology & Immunohistochemistry

One million ADHLSC (≧90% viability) were injected into the spleen of 6-to 14-day-old uPA^(+/+)-SCID mice. Before transplantation, mice showedundetectable serum albumin. Immunohistochemistry in mice liver sampleswas performed on four μm-thick liver sections that were stained withhematoxylin and eosin (HE) for overall histopathological evaluation. Forimmunostaining, liver slides were incubated overnight with primaryantibodies at room temperature. Detection was performed after incubatingthe slices with peroxidase labeled polymer and substrate chromogen(Envision-DAB system, Dako, Carpinteria, Calif.). Counterstaining wasperformed using Hematoxylin.

The transgenic mouse model utilized for the purpose combines a liverpathology (uPA) with immunodeficiency (SCID). After intrasplenictransplantation of the ADHLSC suspension, the uPA⁺⁺-SCID mice were leftto recover for 10 weeks. The analysis of the livers of uPA/SCID micetransplanted with ADHLSC, demonstrated that these cells were able toengraft (FIG. 4) and to differentiate into mature hepatocytes (FIG. 5).Furthermore, human albumin was detected in the serum of thesetransplanted mice 10 weeks post-transplantation whereas the level ofexpressed alpha-fetoprotein, a marker of tumour development, stayeduntraceable.

The transplanted cells did not over-proliferate as shown by microscopicobservation indicating the absence of tumorigenic colonies and normallevel of expression of the tumorigenic markers alpha-fetoprotein andKi67. The normal level of expression corresponds essentially to thelevel of expression measured in normal hepatocytes and less than thelevel of expression measured in the tumorigenic modified human livercell line, e.g., HepG2.

Example 2

An exemplary treatment with the liver originated progenitor or stemcells of the invention, cell lines thereof or cell populationscomprising such (specifically mentioning, albeit of course not limitedto, the LMBP 6452CB line), or progeny thereof, optionally geneticallymodified, may be as follows.

Cells are infused in serial injections, preferably not exceeding 25 to50×10⁶ cell/kg, preferably 4 hours apart, or 8 hrs apart, or more than 8hrs up to one week, or more than one week. A total cell quantity of250×10⁶ cell/kg, or 500×10⁶ cell/kg are infused over few days,preferably one or preferably two weeks. Serial infusions can be repeatedas required, every month, or every six months, or every year or more.

Access to the portal vein is by direct puncture under radiological andor ultrasound guidance, via a puncture needle, or via a percutaneouscatheter, or via a Port-a-cath R device, or via a Broviac R deviceinserted surgically in any vessel draining to the portal vein,preferably the inferior mesenteric vein, or a colonic vein. The cathetercan be left in place for several hours, preferably several days,preferably several weeks, or preferably several months up to two years,or preferably longer for repeating infusions whenever needed.

Immunosuppression is started the day of infusion, preferably the daybefore, preferably with Tacrolimus (FK506) and steroids. Through bloodlevel of Tacrolimus is preferably 8 ng/ml initially, 6 ng/ml after threemonths, 4 ng/ml after 6 months, then kept around 4 ng/ml. Steroids arepreferably given as prednisone or prednisolone, initially 5 mg/kg at day1, 4 mg/kg at day 2, 3 mg/kg at day 3, 2 mg/kg at day 4, 1 mg/kg at day5, and then progressively decreased to reach 0.25 mg/kg at 3 months, andstop at six months. Alternative immunosuppression may include, alone orin combination, Ciclosporin A, anti IL2 receptor antibodies, antithymocyte globulins, or any anti human lymphocyte monoclonal orpolyclonal antibodies, mycophenolate mofetyl or azathioprine or anyantimetabolite agent, ciclosporin, rapamune or any other calcineurininhibitor.

What is claimed is:
 1. An isolated human progenitor or stem celloriginated from human adult liver characterized in that: (a) theisolated human progenitor or stem cell expresses at least themesenchymal markers vimentin and α-smooth muscle actin (ASMA), (b) theisolated human progenitor or stem cell expresses the hepatocyte markeralbumin (ALB), (c) the isolated human progenitor or stem cell isnegative for cytokeratin-19 (CK-19), and (d) the isolated humanprogenitor or stem cell has mesenchymal-like morphology; wherein saidcell is further genetically modified by introduction of a nucleic acidthat encodes gene products which enhance the replicative capacity,growth, differentiation and/or functioning of said cell.
 2. Theisolated, genetically modified human progenitor or stem cell accordingto claim 1, wherein said cell is genetically altered with a nucleic acidencoding a telomerase reverse transcriptase (TERT) of any species. 3.The isolated, genetically modified human progenitor or stem cellaccording to claim 1, wherein said cell is genetically altered with DNAencoding the SV40 large T, by infecting with Epstein Barr Virus,introducing oncogenes, by introducing viral replication genes, or byfusing said cells with an immortalized cell line.
 4. The isolated,genetically modified human progenitor or stem cell according to claim 3,wherein said oncogenes are myc and/or ras.
 5. The isolated, geneticallymodified human progenitor or stem cell according to claim 3, whereinsaid viral replication gene is adenovirus E1a.
 6. The isolated,genetically modified human progenitor or stem cell according to claim 1,wherein said cell is stably or transiently transfected or transformedwith the nucleic acid.
 7. The isolated, genetically modified humanprogenitor or stem cell according to claim 1, wherein said cell ismodified to constitutively or inducibly over-express a polypeptidenormally expressed by hepatocytes.
 8. The isolated, genetically modifiedhuman progenitor or stem cell according to claim 7, wherein said cell ismodified to constitutively or inducibly over-express a polypeptideselected from the group consisting of ornithine transcarbamylase,arginosuccinate synthetase, arigininosuccinate lyase, arginase, carbamylphosphate synthase, N-acetyl glutamate synthase, glutamine synthetase,glycogen synthetase, glucose-6-phosphatase, succinate dehydrogenase,glucokinase, pyruvate kinase, acetyl CoA carboxylase, fatty acidsynthetase, alanine aminotransferase, glutamate dehydrogenase, ferritin,low density lipoprotein (LDL) receptor, P450 enzymes, and/or alcoholdehydrogenase, albumin, transferrin, complement component C3,a2-macroglobulin, fibrinogen, Factor XIII, Factor IX, anda1-antitrypsin.
 9. The isolated, genetically modified human progenitoror stem cell according to claim 1, wherein said cell is modified toconstitutively or inducibly over-express a hormone or an antibody. 10.The isolated, genetically modified human progenitor or stem cellaccording to claim 1, further characterized in that the isolated humanprogenitor or stem cell expresses the markers CD90, CD73, and CD44. 11.The isolated, genetically modified human progenitor or stem cellaccording to claim 1, which further expresses one or more other hepaticor hepatocyte markers selected from the group consisting of CD29,alpha-fetoprotein (AFP), alpha-1 antitrypsin, HNF-4 and MRP2transporter.
 12. The isolated, genetically modified human progenitor orstem cell according to claim 1, which expresses CD29, AFP, alpha-1antitrypsin and MRP2 transporter.
 13. A cell line of or a cellpopulation comprising the isolated, genetically modified human liverprogenitor or stem cell according to claim 1, and progeny thereof thatexpress the mesenchymal markers vimentin and ASMA, express thehepatocyte marker ALB, is negative for CK-19, and has mesenchymal-likemorphology.
 14. The genetically modified cell line or cell population ofclaim 13, or progeny thereof including differentiated progeny thatexpress the mesenchymal markers vimentin and ASMA, express thehepatocyte marker ALB, is negative for CK-19, and has mesenchymal-likemorphology, for use in therapy.
 15. A composition comprising thegenetically modified human liver progenitor or stem cell according toclaim
 1. 16. A pharmaceutical composition comprising the geneticallymodified human liver progenitor or stem cell according to claim 1 and apharmaceutically acceptable carrier.
 17. A method of treating liverdisease comprising administering the genetically modified human liverprogenitor or stem cells according to claim 1 or progeny thereof thatexpress the mesenchymal markers vimentin and ASMA, express thehepatocyte marker ALB, is negative for CK-19, and has mesenchymal-likemorphology to an individual in need thereof.
 18. The A method oftreating liver disease according to claim 17, wherein the cells areinfused via the portal vein, by direct hepatic injection, or byintrasplenic injection.
 19. The pharmaceutical composition according toclaim 16, wherein the cells are provided as a cell suspension.
 20. Amethod for enhancing the regeneration of an injured or diseased livercomprising administering the genetically modified human liver progenitoror stem cell according to claim 1 or progeny thereof that express themesenchymal markers vimentin and ASMA, express the hepatocyte markerALB, is negative for CK-19, and has mesenchymal-like morphology to anindividual in need thereof.
 21. The method for enhancing theregeneration of an injured or diseased liver according to claim 20,wherein the cells are infused via the portal vein, by direct hepaticinjection, or by intrasplenic injection.